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Electrostatic and structural investigations into the xylanase Cex from Cellulomonas fimi Poon, David Kai Yuen 2007

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1 Electrostatic and Structural Investigations into the Xylanase Cex from Cellulomonas fimi by DAVID KAL YUEN POON B.Sc., The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY m THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA September 2007 © David Kai Yuen Poon, 2007 Abstract i ABSTRACT The overall goal of this thesis was to study the Cellulomonas fltni family 10 P-1,4-xylanase Cex along its retaining double-displacement reaction pathway using NMR spectroscopy, complimented by X-ray crystallography and enzyme kinetics. NMR relaxation measurements demonstrated that the backbone and tryptophan sidechains of Cex are well ordered on the ns-ps and ms-ps timescales in both its free and glycosyl-enzyme intermediate forms. However, the local and global stability of Cex increased upon glycosyl-enzyme intermediate formation. The relatively rigid active site may be necessary to bind, distort, and subsequently hydrolyze target glycosides, as well as to maintain the lifespan of the secreted xylanase in the extracellular milieu. Cex is a modular enzyme composed of a xylan-specific catalytic domain with a cellulose-binding domain, joined by a proline-threonine linker. Using NMR spectroscopy, it was demonstrated that the domains do not measurably interact with each other, and that the proline-threonine linker tethering them is predominantly unstructured and conformationally flexible on the sub-ns timescale. This supports a "beads-on-a-string" model of Cex in which it is anchored to cellulose via its cellulose-binding domain, yet cleaving nearby xylan by virtue of the flexible glycosylated linker. Substrate binding and catalysis by Cex are controlled by an intricate network of charges and hydrogen bonding interactions within its active site. Using a plethora of isotopic labelling strategies and NMR-monitored pH titrations, the charge states and/or pKa values of all the ionizable groups (Lys, His, GIu, and Asp) in the Cex active site were determined in both its free and inhibited forms. Most importantly, the pKa of the general acid/base residue Glu 127 was shown to cycle to serve its role as both a general acid (pKa 7.4) and a general base (pKa 4.2) in the glycosylation and the deglycosylation steps of the P-retaining double-displacement reaction, respectively. Abstract ii The family 10 xylanases display significant topological variations in the number of binding cleft substrate recognition sites. Two xylotriose-based inhibitors of Cex were examined by X-ray crystallography and enzyme inactivation kinetics, and these studies revealed that unlike other family 10 xylanases, Cex does not contain a significant -3 glycone binding subsite. Together, this research provides a unique perspective of the synergistic interactions between dynamics, electrostatics, and global architecture that govern the function of the multi-domain xylanase Cex. Table of Contents i i i TABLE OF CONTENTS Abstract i Table of Contents • iii List of Tables "x List of Figures ••••• x List of Schemes xiv List of Abbreviations xv List of Amino Acid Abbreviations • xix Acknowledgements xx Co-Authorship Statement • xxi Chapter 1 1 1.1 Carbohydrates 2 1.2 Glycoside Hydrolases 4 1.2.1 Classifications of glycoside hydrolases :. : 4 1.2.2 Mechanisms of glycoside hydrolases 5 1.2.3 Xylan and xylanases 8 1.2.3.1 Cex / CfXynlOA xylanase from Cellulomonasfimi ....10 1.3 Carbohydrate-Binding Modules -.. 17 1.3.1 Classification of carbohydrate-binding modules 17 1.3.2 CBM binding and polysaccharide hydrolysis .....17 1.3.2 The family 2 CBM from Cex 18 1.4 Specific Aims of the Thesis 19 Chapter 2 ; 21 2.1 Introduction • 22 Table of Contents iv 2.2 Materials and Methods 24 2.2.1 Protein expression and purification 24 2.2.2 NMR spectral assignment 26 2.2.3 Assignment validation by secondary structure prediction 26 2.2.4 Chemical shift perturbations to map structural changes 27 2.2.5 1 5 N relaxation measurements 27 2.2.6 Proton-deuterium exchange 30 2.2.7 Thermal denaturation measurements 31 2.2.8 Thermolysin-catalyzed proteolysis -.32 2.3 Results 33 2.3.1 NMR spectral assignments 33 2.3.2 Secondary structure analysis 36 2.3.3 Chemical shift perturbations upon glycosyl-enzyme intermediate formation ; 36 2.3.4 Backbone amide dynamics from 1 5 N relaxation measurements 40 2.3.5 Tryptophan sidechain dynamics from 1 5 N relaxation measurements 45 2.3.6 Amide and indole hydrogen exchange 45 2.3.7 Thermal denaturation 49 2.3.8 Thermolysin-catalyzed proteolysis 49 2.4 Discussion 54 2.4.1 NMR spectral assignments and structural analyses 54 2.4.2 Backbone amide and tryptophan indole dynamics 55 2.4.3 Stabilization of CexCD by glycosyl-enzyme intermediate formation 56 2.4.4 Implications for catalysis 59 Table of Contents V 2.5 Conclusion 61 Chapter 3 62 3.1 Introduction 63 3.2 Materials and Methods 66 3.2.1 Expression and purification of uniformly 15N-labelled Cex, CexCD, andCexCBD 66 3.2.2 Expression of 1 5 N Thr- labelled Cex 66 3.2.3 Expression of glycosylated 1 5 N Thr-labelled Cex 66 3.2.4 NMR spectroscopy 67 3.3 Results 69 3.3.1 Non-glycosylated Cex expressed from is. coli 69 3.3.1.1 Structural independence between the catalytic and cellulose-binding domains 69 3.3.1.2 Interdomain mobility between the catalytic and cellulose-binding domains 73 3.3.1.3 Conformationally dynamic and unstructured PT-Linker 74 3.3.2 Glycosylated Cex expressed from S. lividans 79 3.3.2.1 Production of glycosylated Cex 79 3.3.2.2 Independence of the catalytic and cellulose-binding domains with PT-Linker glycosylation 79 3.3.2.3 Conformationally dynamic and predominantly unstructured glycosylated PT-Linker .- 81 3.4 Discussion 83 3.4.1 Cex is composed of independent catalytic and cellulose-binding domains tethered by a flexible PT-Linker 83 Table of Contents y i 3.4.2 Glycosylation partially dampens the fast timescale motions of the PT-linker, but does not perturb the catalytic or cellulose-binding domains 84 3.4.3 Implications for the structure of Cex 85 3.4.4 Implications for catalysis by Cex 87 3.5 Conclusion 89 Chapter 4 90 4.1 Introduction 91 4.1.1 Electrostatic interactions in proteins ....91 4.1.2 The family 10 xylanase Cex ...97 4.1.3 NMR spectroscopy and pKa determination 102 4.2 Materials and Methods 104 4.2.1 Expression and purification of uniformly and selectively isotopic-labelled CexCD.... : 104 4.2.2 NMR spectroscopy , 104 4.2.3 Ionization states and pH titrations of CexCD by NMR spectroscopy 105 4.3 Results 110 4.3.1 Lysines... '. ....110 4.3.1.1 Lysine NMR assignments 110 4.3.1.2 Lysine ionizations and pH-dependent titrations 110 4.3.2 Histidines 117 4.3.2.1 Histidine NMR assignments '. 117 4.3.2.2 Histidine ionization states and pKa measurements 117 4.3.3 Glutamic and aspartic acids 120 4.3.3.1 Carboxyl NMR assignments 120 ' Table of Contents vii 4.3.3.2 Glutamic and aspartic acid ionization states and pKa measurements , 125 4.3.3.2.1 Direct Glu 1 3 C 8 titration 125 4.3.3.2.2 Through bond detected 1 3 C 8 titrations 125 4.3.3.2.3 Ionization information from 1 3 C 5 and 1 3 C Y chemical shifts 127 4.3.3.2.4 'H-^N monitored titrations 129 4.3.3.2.4.1 Catalytic Nucleophile (Glu233) and the Catalytic General Acid/Base (Glu 127) : 133 4.3.3.2.4.2 A substrate-binding active-site glutamate (Glu43) 139 4.3.3.2.4.3 An ionization modulator (Asp235) 139 4.4 Discussion 145 4.4.1 Lysines 145 4.4.2 Non-active site histidines (His 85, Hisl07, Hisl 14) 147 4.4.3 Interactions between Aspl23, His80, and Glu233 148 4.4.4 Interactions between Glu233, His205, and Asp235 152 4.4.5 Interplay between the nucleophile Glu233 and the general acid/base Glul27 : ....154 4.5 Conclusion......... , 156 Chapter 5 159 5.1 Introduction 160 5.1.1 The family 10 xylanase Cex 163 5.2 Materials and Methods 167 5.2.1 Protein expression and purification ...167 5.2.2 Synthetic mechanism-based inhibitors .• 167 5.2.3 Crystallization of the CexCD-inhibitor complexes......... 167 Table of Contents . viii 5.2.4 Kinetic inactivation measurements 168 5.3 Results.. 168 5.3.1 CexCD inactivation kinetics 168 5.3.2 X-ray crystal structures of inhibitor-enzyme complexes , 169 5.4 Discussion 176 5.4.1 The lack of a -3 glycone binding subsite in CexCD 176 5.4.2 Comparison of the Cex binding site topology with those of other family 10 xylanases 179 5.4.2.1 The glycone (-) binding region 180 5.4.2.2 The aglycone (+) binding region 183 5.5 Conclusion 186 References. 187 Appendix 1 205 Appendix 2 213 Appendix 3 221 Appendix 4 ; : 228 List of Tables ix LIST OF TABLES Table 4.1. Random-coil pKa values of proteins 92 Table 4.2. Labelling strategies and NMR experiments used to assign resonances of ionizable groups in the Cex active site 106 Table 4.3. Labelling and pH titration strategies used to determine pKa values of ionizable groups in apo- and 2FCb-CexCD 109 Table 4.4. Summary of the charge state and pKa values limits of the two observable Lys residues in apo- and 2FCb-CexCD 116 Table 4.5. Summary of charge/tautomer states and pKa values of the His residues in apo- and 2FCb-CexCD 122 Table 4.6. 1 3 C 8 chemical shifts and possible charge states of the glutamic acid residues in apo- and 2FCb-CexCD 130 Table 4.7. 1 3 C y chemical shifts and possible charge states of the glutamic acid residues in apo- and 2FCb-CexCD 131 Table 4.8. Summary of the charge states and pKa values of the Glu residues in apo-and 2FCb-CexCD , 138 Table 4.9. Summary of the charge states and pKa values of the Glu residues in apo-and 2FCb-CexCD 144 Table 5.1. Kinetic parameters for selected mono- and di-saccharide substrates and inhibitors used to probe the active site of Cex 164 Table 5.2. Summary of kj and Kj results for CexCD 171 Table 5.3. The crystal refinement statistics for 2FOX3-CexCD and 2FSX3-CexCD 174 List of Figures X LIST OF FIGURES Figure 1.1. Retaining mechanism of a B-glycosidase 6 Figure 1.2. Inverting mechanism of a p-glycosidase 7 Figure 1.3. Molecular structure of a generic xylan polymer 9 Figure 1.4. The modular architecture of Cex 11 Figure 1.5. X-ray crystal structure of the Cex catalytic domain 12 Figure 1.6. Commonly used synthetic aryl substrates 14 Figure 1.7. pH-dependence of Cex towards 2,4-DNPC 15 Figure 2.1. 'H- 1 5N TROSY-HSQC spectra of apo-CexCD 34 Figure 2.2. ! H- 1 5 N TROSY-HSQC spectra of 2FCb-CexCD 35 Figure 2.3. A summary of the HX, shift perturbation mapping, and secondary structure prediction.. 37 Figure 2.4. Chemical shift perturbations of CexCD upon covalent modification 39 Figure 2.5. 1 5 N T,, T 2 , NOE, and S2 for apo-CexCD 41 Figure 2.6. 1 5 N T,, T 2 , NOE, and S2 for apo-CexCD 42 Figure 2.7. Quantitative HX comparison between apo- and 2FCb-CexCD 47 Figure 2.8. Qualitative comparison of HX between apo- and 2FCb-CexCD for selected active site residues 48 Figure 2.9. Covalent modification selectively protects active site amides from HX 50 Figure 2.10. CD-monitored thermal denaturation of apo- and 2FCb-CexCD 51 Figure 2.11. Proteolytic degradation of apo- and 2FCb-CexCD by thermolysin 53 Figure 3.1. 'H- 1 5 N TROSY-HSQC spectra for CexCD, CexCBD, and Cex 70 Figure 3.2. Amide chemical shift perturbation between non-glycosylated Cex and the isolated CexCD and CexCBD 71 Figure 3.3. Amide chemical shift perturbation between non-glycosylated Cex and the in situ separated CexCD and CexCBD 72 List of Figures ' xi Figure 3.4. Amide ' D N H RDC measurements for the catalytic domain, cellulose-binding domain, and the PT-linker region of non-glycosylated Cex 75 Figure 3.5. 'H- 1 5 N TROSY-HSQC spectra of 15N-Thr-labelled non-glycosylated and glycosylated Cex 76 Figure 3.6. Heteronuclear 'H-I^N} NOE measurements of 15N-Thr-labelled non-glycosylated and glycosylated Cex 78 Figure 3.7. Amide chemical shift perturbation between 15N-Thr-labelled non-glycosylated and glycosylated Cex 80 Figure 4.1. The family 11 xylanase Bex and pKa cycling 94 Figure 4.2. Inter-residue and inter-molecular active site interactions in apo- and 2FCb-CexCD 99 Figure 4.3. pH-dependence of wild-type and E233D mutant of Cex towards 2,4-DNPC 101 Figure 4.4. NMR experiments used to assign resonances of ionizable groups in the Cex active site 107 Figure 4.5. 'H- 1 5N HMQC spectra of apo- and 2FCb-CexCD at varying pH and temperature conditions ....Ill Figure 4.6. 'H- 1 5 N HMQC spectra of 2FCb-CexCD, Xblso-CexCD, and Xblm-CexCD 112 Figure 4.7. ! H ? - and '^-observed pH titrations of Lys47 and Lys302 in apo- and 2FCb-CexCD 114 Figure 4.8. Assignment of the protonation and tautomerization states of the histidines in apo- and 2FCb-CexCD 118 Figure 4.9. , 3C-CPMG-HSQC spectra of 13CE,-His-labelled apo-, 2FCb, Xblso-, and Xblm-CexCD 119 Figure 4.10. ' H E l - and 13CEl-monitored pH titrations of 1 3C e l-His labelled apo- and 2FCb-CexCD 121 List of Figures x i i Figure 4.11. 1 3 C 8 / y assignments of carboxyls in apo- and 2FCb-CexCD using C 8 / Y(C pC aC')NH experiments '. 124 Figure 4.12. 1-D 13C5-monitored pH titration of apo-CexCD 126 Figure 4.13. Distribution of 1 3 C 8 / y chemical shifts in apo- and 2FCb-CexCD 128 Figure 4.14. 'H- 1 5N NMR reporter groups used to probe for the pH titrations of Glu23 3 and Glu 127 in apo- and 2FCb-CexCD 134 Figure 4.15. pH-dependent chemical shift changes of Trp273 1Be\ His80 1 3 C E l , and Trp84 'H E l in apo- and 2FCb-CexCD ' 135 Figure 4.16. pH-dependent chemical shift changes of Tyrl71 1 5 N H in apo- and 2FCb-CexCD to probe for the pH titrations of Glul27 and Aspl70 '. 137 Figure 4.17. pH-dependent chemical shift changes of Glu43 ' H N in apo- and 2FCb-CexCD to probe for the pH titrations of Glu43 140 Figure 4.18. 'H-^N NMR reporter groups used to probe for the pH titrations of Asp235 in apo- and 2FCb-CexCD 142 Figure 4.19. pH-dependent chemical shift changes of His205 I 3 C e l , Trp281 1 H N , and Trp273 'H E l in apo- and 2FCb-CexCD 143 Figure 4.20. Stabilization of the positively-charged Lys302 146 Figure 4.21. Inter-residue interactions between Aspl23, His80, and Glu233 149 Figure 4.22. pH-dependence of wild-type and H80A Cex towards PNPX2 151 Figure 4.23. Inter-residue interactions between Glu233, His205, and Asp235 153 Figure 4.24. Inter-residue and inter-molecular interactions and pKa values of the active site ionizable groups in apo- and 2FCb-CexCD 157 Figure 5.1. Cex surface binding cleft : 161 Figure 5.2. Subsite nomenclature 162 Figure 5.3. Synthetic inhibitors of Cex 166 List of Figures x i i i Figure 5.4. Cex inactivation kinetics 170 Figure 5.5. X-ray crystal structure of 2FOX3-CexCD 172 Figure 5.6. X-ray crystal structure of 2FSX3-CexCD 173 Figure 5.7. X-ray crystal structures of 2FOX3-CexCD and 2FSX3-CexCD highlighting the electron densities of the two bound inhibitors 175 Figure 5.8. Potential hydrolysis pathways of the xylotriose-derived inhibitor 2FO-DNPX3 178 Figure 5.9. Position of Trp281 in 2FX2-CexCD and 2FCb-CexCD 181 Figure 5.10. Bound DNP leaving group in the X-ray crystal structure of 2FOX3-CexCD 184 List of Schemes ' x iv I",- • LIST OF SCHEMES Scheme 2.1. Hydrogen exchange in proteins 30 Scheme 4.1. Acid-base equilibrium 91 List of Abbreviations XV LIST OF ABBREVIATIONS Aco £280nm 2,4-DNPC 2,4-DNPG 2,4-DNPX2 2FCb-CexCD 2F-DNPC 2F-DNPX2 2FO-DNPX3 2FOX3-CexCD 2FS-DNPX3 2FSX3-CexCD 2FX2-CexCD 4-O-MeGlcA apo-CexCD Bex Change in frequency Molar absorptivity at 280 nm Effective correlation time Internal correlation time Time constant describing fast internal motions Global tumbling time Time constant describing slow internal motions 2,4-Dinitrophenyl P-cellobioside 2,4-Dinitrophenyl glucopyranoside 2,4-Dinitrophenyl p-xylobioside CexCD covalently-inhibited with 2-fluoro-cellobioside 2,4-Dinitrophenyl 2-deoxy-2-fluoro-p-cellobioside 2,4-Dinitrophenyl 2-deoxy-2-fluoro-P-xylobioside 2,4-Dinitrophenyl 2-deoxy-2-fluoro-P-xylotrioside CexCD covalently-inhibited with 2-deoxy-2-fluoro-P-xylotrioside 2,4-Dinitrophenyl 2-deoxy-2fluoro-4n-thio-P-xylotrioside CexCD covalently-inhibited with 2-deoxy-2fluoro-4I1-thio-P-xylotrioside CexCD covalently-inhibited with 2-fluoro-xylobioside 4-O-methyl-D-glucuronic acid Free form of CexCD without bound inhibitors Bacillus circulans family 11 xylanase List of Abbreviations xvi B-value X-ray crystallographic thermal factor CBD Cellulose-binding domain CBM Carbohydrate-binding module CD Circular dichroism spectroscopy Cex Cellulomonas fimi xylanase CexCBD . Cellulose-binding domain of Cex (residues 336 to 443) CexCD Catalytic domain of Cex (residues 1 to 315) CPMG Carr-Purcell-Meiboom-Gill DCN-labelled 2 H/ 1 3 C/ 1 5 N Isotopically-labelled Dmax Maximum dimension of the conformational ensemble detected by SAXS DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DNP Dinitrophenyl group EC Enzyme Commission (Classification number) of the International Union of Biochemistry EDTA Ethylenediamine tetraacetic acid ESI-MS Electrospray ionization mass spectrometry FID Free induction decay FPLC Fast protein liquid chromatography GH Glycoside hydrolase HMQC Heteronuclear multiple quantum correlation HSQC Heteronuclear single quantum correlation HX Hydrogen exchange IPTG Isopropyl P-D-thioglucopyranoside List of Abbreviations xv i i J(co) Spectral density function K a Acid equilibrium constant kcat Catalytic rate constant (turnover number) ke X Observed proton-deuterium exchange rate K; Equilibrium inhibitor binding constant k, Inactivation rate constant K m Michaelis constant kobs Apparent rate constant LB Luriabroth MALDI-TOF Matrix-assisted laser desorption ionization time-of-flight MWCO Molecular weight cut-off NAD + ( 0 X) P-nicotinamide adenine dinucleotide NADH(red) P-nicotinamide adenine dinucleotide phosphate NCTC National Collection of Type Cultures NMR Nuclear magnetic resonance spectroscopy NOE Nuclear Overhauser effect OD60o Optical density (or absorbance) at 600 nm ONPX2 o-Nitrophenyl-p-D-xylobioside ORF Open reading frame PEG Polyethylene glycol pH* Measured pH without correction for isotope effects PNPC /7-Nitrophenyl P-cellobioside PNPX /7-Nitrophenyl xylopyranoside PNPX2 p-Nitrophenyl P-xylobioside ppm Parts per million List of Abbreviations xviii PT-linker Proline/threonine linker j oining CexCD with CexCBD (residues 316 to 335) RDC Residual dipolar coupling RDS Rate determining step ReX Conformational exchange rate rmsd Root mean square deviation RNA Ribonucleic acid S2 General order parameter SAXS Small-angle X-ray scattering SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis S f2 Order parameter corresponding to fast internal motions S s2 Order parameter corresponding to slow internal motions Ti Longitudinal relaxation time t1/2 Half-life T 2 Transverse relaxation time T m Midpoint unfolding temperature Tris 2-amino-2-hydroxymethyl-1,3 -propanediol TROSY Transverse-relaxation optimized spectroscopy WT Wild type Xblm Xylobio-imidazole Xblm-CexCD CexCD non-covalently complexed with xylobio-imidazole Xblso Xylobio-isofagomine Xblso-CexCD CexCD non-covalently complexed with xylobio-isofagomine List of Amino Acid Abbreviations xix LIST OF AMINO ACID ABBREVIATIONS A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I He Isoleucine K Lys Lysine L Leu Leusine M Met Methionine N Asn Asparagine P Pro Proline Q Gin Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine w Trp Tryptophan Y Tyr Tyrosine Asx or Glx Aspartic acid & Asparagine or Glutamic acid & Glutamine Acknowledgements xx ACKNOWLEDGEMENTS My sincerest gratitude goes to my research supervisors, Drs. Lawrence P. Mcintosh and Stephen G. Withers, for their insightful guidance and constant support during the course of my graduate dissertation. Their relentless drive and dedication towards their respective laboratories, and their constant thirst for the understanding of biological and chemical processes around us, are both humbling and inspiring. Under their tutelage, I have grown as a person and as a scientist, and for that I will always be grateful. I am forever indebted to my wife Katherine, who has been my pillar of strength and determination since my freshman year at UBC. My accomplishments and successes would not have been possible without her encouragements and steadfast support throughout times of frustrations and achievements that are part of the research process. To my Mom and Dad, your foresight in immigrating to Canada, thus allowing me the opportunity to grow and thrive in Vancouver, is invaluable. Thank you also for your patience and hard work in raising me into who I am today. Also, my heartfelt appreciation goes to the present and former members of the Mcintosh and Withers groups. With special thanks to Drs. Greg Lee, Mark Okon, and Mario Schubert for their endless advice in all matters relating to NMR spectroscopy and computational software; Emily Kwan, and Drs. Martin Ludwiczek and Jacqueline Wicki for their guidance in protein biochemistry and enzymology; Dr. Igor D'Angelo for his expertise in X-ray crystallography; Terrence Kantner for synthesizing the xylotriose inhibitors; and Miranda Joyce for her assistance with navigating through university bureaucratic hurdles. Co-Authorship Statement xxi CO-AUTHORSHIP STATEMENT The majority of the results from Chapter 2 were presented in the peer-reviewed journal Biochemistry (Poon, D. K. Y., Ludwiczek, M. L., Schubert, M., Kwan, E. M., Withers, S. G., and Mcintosh, L. P. (2007) NMR spectroscopic characterization of a P-(l,4)-glycosidase along its reaction pathway: stabilization upon formation of the glycosyl-enzyme intermediate, Biochemistry 46, 1759-1770.) (Poon et al, 2007a) (© American Chemical Society, 2007 by permission). The NMR spectral assignment of the tryptophan indoles ('HE l, and 1 5NE l) was performed by Dr. Mario Schubert, and the proteolytic degradation of apo- and 2FCb-CexCD using thermolysin was measured by Dr. Martin L. Ludwiczek. Emily M. Kwan expressed and purified the 2H/13C/15N-labelled CexCD. The results discussed in Chapter 3 have been published in the peer-reviewed periodical Journal of Biological Chemistry (Poon, D. K. Y., Withers, S. G., and Mcintosh, L. P. (2007) Direct demonstration of the flexibility of the glycosylated proline-threonine linker in Cellulomonas fimi xylanase Cex through NMR spectroscopic analysis, J. Biol. Chem. 282, 2091-2100.) (Poon et al, 2007b) (© Journal of Biological Chemistry, 2007 with permission). Dr. Lawrence Mcintosh performed the data analysis pertaining to the residual dipolar coupling analysis of the full-length glycosylated Cex. The work pertaining to the CexCD lysines in Chapter 4 was reported in the peer-reviewed periodical Journal of the American Chemical Society (Poon, D. K. Y., Schubert, M., Au, J., Okon, M.j Withers, S. G., and Mcintosh, L. P. (2006) Unambiguous determination of the ionization state of a glycoside hydrolase active site lysine by 'H-^N heteronuclear correlation spectroscopy, J. Am. Chem. Soc. 128, 15388-15389.) (Poon et al, 2006) (© American Chemical Society, 2006 with permission). Resonance assignment of Lys302 was performed by Dr. Mark Okon. The expression of the 15N-labelled K47A Cex and the pH titrations of apo-CexCD and 2FCb-CexCD were accomplished with the assistance of Jason Au. Figures 4.5, 4.6, and 4.7 were adapted from the above published work. Co-Authorship Statement xx i i The study related to the CexCD histidines in Chapter 4 was reported in the peer-reviewed periodical Biochemistry (Schubert, M., Poon, D. K. Y., Wicki, J.', Tarling, C. A., Kwan, E. M., r Nielsen, J. E., Withers, S. G., and Mcintosh, L. P. (2007) Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy, Biochemistry 46, 7383-7395.) (Schubert et al., 2007) (© American Chemical Society, 2007 with permission). The majority of the work in this publication was carried out by Dr. Mario Schubert. My specific contributions included the expression and purification of the isotopically-labelled CexCD samples, and the pH titrations of apo- and 2FCb-CexCD. The kinetic study of H80A Cex was performed by Dr. Jacqueline Wicki. Data from this publication were adapted in this chapter as Figures 4.8, 4.9, and 4.10 and Table 4.5 (©American Chemical Society, 2007 adapted with permission). The work presented in Chapter 5 was performed in collaboration with Terrence Kantner and with Dr. Igor D'Angelo at the University of British Columbia. Mr. Kantner synthesized 2FO-DNPX3 and 2FS-DNPX3, while Dr. D'Angelo collected the X-ray diffraction data and refined the crystallographic structures of CexCD covalently modified with these inhibitors. A manuscript summarizing these findings in combination with similar data collected for the family 11 xylanase Bex is in preparation. Chapter 1 - General Introduction 1 Chapter 1 General Introduction Chapter 1 - General Introduction 2 1.1 CARBOHYDRATES Carbohydrates account for the majority of the carbon in the biosphere. The traditional view of these polysaccharide polymers is that their stability in water renders them suitable as media for the storage of metabolic energy in their a-linked amylose and glycogen forms and as structural supports in their B-linked cellulose and chitin forms (Wolfenden et al, 1998). Compared to other biological polymers, they are very stable under neutral, aqueous conditions: glycosidic bonds have spontaneous hydrolytic half-lives 30 times and 10,000 times longer than phosphodiester (DNA and RNA) and peptide (protein) bonds, respectively. For cellulose, this equates to a half-life of approximately five million years (Wolfenden et al, 1998). Carbohydrates, in the forms of glycoproteins, proteoglycans, glycolipids, and polysaccharides play pivotal roles in cell physiology and in the development of microbes, plants, and animals (Davies et ai, 2005). This is, in part, due to their structural diversity relative to DNA, RNA, and proteins. For example, in generic hexanucleotides and hexapeptides, 4096 and 6.4 xlO7 isoforms are possible, respectively, whereas a reducing hexasaccharide has 1012 possible isoforms (Laine 1994). With the continual acquisition of genome sequences, it has been found that carbohydrate-active enzymes, which catalyze the synthesis and breakdown of glycosidic bonds, account for 1-3% of the proteins encoded by most organisms. Over 12,000 glycosyltransferase and glycoside hydrolase open reading frames (ORFs) have been identified to date. The development and function of physiological systems, including protein-protein and cell-cell interactions, appear to be modulated by glycans, which in turn are regulated by these carbohydrate-active enzymes. Most of the glycosyltransferases and glycoside hydrolases reside in the Golgi apparatus of eukaryotes, and are responsible for constructing the glycan repertoire that is found on cell surfaces. Increasingly, dysregulation of glycan synthesis, which is often due to the over- or under-expression of modifying enzymes, is recognized as the etiology for a growing number of human genetic diseases (Lowe and Marth, 2003). Humans are also symbiotically dependent on three gastrointestinal tract bacteria, Chapter 1 - General Introduction. 3 Bacteroides thetaiotaomicron, Bacteroides fragilis N C T C 9343, and Bifidobacterium longum, for which the percentage of their genomes devoted to carbohydrate-active enzymes are amongst the highest of all systems studied at 6.6%, 4.8%, and 3.6%, respectively (Davies et al, 2005). \ Chapter 1 - General Introduction 4 1.2 GLYCOSIDE HYDROLASES 1.2.1 Classifications of glycoside hydrolases Glycoside hydrolases, or glycosidases, can be generally classified based on their specificity for substrates and on the anomeric stereochemistries of both the substrates and products. Some of these enzymes are highly selective, whereas others are able to catalyze the hydrolysis of a range of glycosides (Gilkes et al, 1991a; Notenboom et al, 1998b). They are thus best named according to the class of substrates that is cleaved with the highest k c a t / K m , where k c a t represents the rate of the slowest step in the enzyme mechanism, while K m is the binding constant between the enzyme and the substrate. Together, k c a t / K m is a second order rate constant describing the events leading up to the first irreversible step in mechanism. Glycoside hydrolases are further designated as a- or p-glycosidases depending on their substrate preference. Finally, they are classified as inverters or retainers based upon the stereochemical outcome of the reaction they catalyze. Retaining glycosidases release glycone products with the same anomeric configuration as the reactants, while inverting glycosidases release products of the opposite anomeric configuration. So for a P-retaining xylanase, the enzyme cleaves P-glycosidic linkages in xylan to produce products with a P-configuration at the newly generated reducing ends. A sequence-based approach to the classification of glycosidases was developed by Dr. Bernard Henrissat (www.CaZy.org). Using this approach, glycoside hydrolases have been organized into 111 distinct "families." With a direct relationship between sequence and fold, such a classification system reflects structural similarities and helps to reveal the evolutionary relationships between distinct families. Remarkably, this also provides a reliable method to derive mechanistic information for these enzymes (Henrissat, 1991; Henrissat and Bairoch, 1993). Often, protein folds are better conserved than their sequences, and with the increasing availability of NMR solution and X-ray crystallographic structures, these glycoside hydrolase families can be further organized into Chapter 1 - General Introduction 5 "clans" (GH-A to -N). Clan classifications are based upon fold conservation, catalytic machinery (types of catalytic residues and positions in the active site), catalytic mechanism (inverting or retaining), and stereochemistry of the substrate (axial or equatorial glycosidic bond) (Henrissat and Bairoch, 1993). Such an organization is advantageous, especially when new sequences are found to be related to more than one family or when structural determinations demonstrate the resemblance between members of different families (Henrissat and Bairoch, 1996). 1.2.2 Mechanisms of glycoside hydrolases Although originally defined using sequences, it is apparent that all members belonging to a given glycoside hydrolase family perform catalysis via the same mechanism (Henrissat, 1998). The majority of glycosidases catalyze the hydrolysis of glycosidic bonds with either a retaining (Figure 1.1) or an inverting (Figure 1.2) mechanism, depending on the stereochemical outcome (Koshland, 1953; Sinnot, 1990). Both types of reaction mechanisms take advantage of general acid/base catalysis, but with a strikingly different arrangement of structural elements - namely the distance between the two catalytic carboxylates (Davies and Henrissat,. 1995; McCarter and Withers, 1994). The average distance is ~ 5.5 A in retaining glycosidases, whereas it ranges from 6-11 A in inverting enzymes. The greater separation for the latter is necessary to accommodate a water molecule, which acts as the displacing nucleophile in a single-step reaction. Classic examples of retaining glycosidases include the P-galactosidase from E. coli (Jacobson et al, 1994), and the xylanases Cex from Cellulomonas fimi (Tull et al, 1991) and Bex from Bacillus circulans (Wakarchuk et al, 1994). Examples of inverting glycosidases include the P-amylase (Mikami et al, 1993) from soybeans and bacteriophage T4 lysozyme (Kuroki et al, 1995). Although the modification or engineering of active site residues offers the potential to change the stereochemical outcomes of the reactions catalyzed by these enzymes, this proves difficult in practice. Using the retaining xylanase Bex, the inter-residue distance between the two catalytic residues was increased ~ 1 A by replacing the nucleophilic glutamate with an aspartate. This did not result in a change in mechanism, but simply, a 10-fold Chapter 1 - General Introduction 6 General Acid - H O ^ O o<®^o Nucleophile General Acid i/WW* H O ^ O OH Nucleophile 5 - / X 0 X) X ^ -R0H General Base - / o \ OH K \ I ) a H Ox . 0 Glycosyl-Enzyme Intermediate or x> 5+ : 8+ H H 0 ^ ^ ° y i H OH v° Figure 1.1. Retaining mechanism of a (3-gtycosidase. The proximal sugar of the glycone is shown with the R-group representing the aglycone. Chapter 1 - General Introduction 7 Figure 1.2. Inverting mechanism of a P-glycosidase. The proximal sugar of the glycone is shown with the R-group representing the aglycone. Chapter 1 - General Introduction 8 crippling of the normal retaining mechanism (Lawson et al, 1996). However, the converse approach of generating a retaining enzyme from an inverting one was successfully performed on T4 lysozyme by the introduction of a new catalytic imidazole in place of an existing non-catalytic threonine (Kuroki etal, 1995). Although prevalent, not all glycoside hydrolases follow the above reaction mechanisms. Recent studies uncovered an example of a "non-typical" reaction mechanism in the family 4 P-glycosidase from Thermotoga maritima. Making use of the redox potential offered by the co-factor NADYNADH, the substrate's C3 is oxidized to a ketone, which effectively acidifies the C2 proton, facilitating its abstraction in a 2,1 elimination of the aglycone. The sequential 1,2 addition of water, followed by re-reduction of the ketone yield the product sugar, with net retention of anomeric configuration (Rajan et al, 2004; Yip et al, 2004). 1.2.3 Xylan and xylanases The plant cell wall represents the largest reservoir of organic carbon in the biosphere with over 1011 tons synthesized annually. The degradation of the cell wall by microbial enzymes represents a key biological process that is central to herbivore nutrition, host invasion by phytopathogenic fungi and bacteria, and the carbon cycle (Coughlan 1985; Gilbert and Hazlewood, 1993; Vardakou et al, 2005). Xylan, a major component of hemicellulose, is a biopolymer comprising of a backbone of xylopyranose rings joined together via P-l,4-glycosidic linkages. Depending on the plant species, this xylan backbone can be decorated to various levels with a variety of modifications. Most commonly, arabinofuranose, acetyl, and 4-methyl-D-glucuronic acid (4-O-MeGlcA) are attached as side chains at the 02 and/or 03 positions of the backbone (Figure 1.3) (Pell et al, 2004a). Although the degradation of the xylan backbone by microorganisms is primarily mediated by glycoside hydrolase family 10 and 11 endo-P-l,4-xylanases, microorganisms typically secrete a battery of different enzymes into the extracellular milieu. Cooperatively, with membrane-attached ct-glucuronidases and Chapter 1 - General Introduction 9 Figure 1.3. Molecular structure of a generic xylan polymer. The generic xylan polymer is decorated with 4-methyl-D-glucuronic acid (4-O-MeGlcA) (red), acetyl (purple), and arabinofuranose (blue). Chapter 1 - General Introduction 10 arabinofuranosidases, the collection of hemicellulolytic enzymes help to break down the long decorated polysaccharide chains into smaller oligosaccharides, which microorganisms can import for use as energy sources (Pell et al, 2004a). Industrial processes also make use of xylanases. For example, the bleaching of pulp in the pulp and paper industry requires the oxidation of lignin, which is responsible for the often undesirable brown "woody" colour. Traditionally, this bleaching process requires the use of caustics and C102 under elevated temperatures. By using xylanases to treat brown pulp, lignin can be removed using less chemicals under milder conditions, thereby making the process more environmentally and financially advantageous. Recent trends in the production of bio-ethanol have spurred renewal of interest in optimizing the activities of enzymes capable of cell wall degradation to produce oligosaccharides and monosaccharides compatible with the fermentation processes being developed. The potential scope of utilizing these enzymes in the energy industry is staggering: the estimated energy content of sugars released annually from natural cell wall degradation is equivalent to 640 billion barrels of oil per annum (Vardakou et al., 2005)! 1.2.3.1 Cex / CfXynlOA xylanase from Cellulomonas fimi The xylanase Cex (C/XynlOA) (EC 3.2.1.8) from Cellulomonas fimi is a secreted enzyme composed of an N-terminal catalytic domain (White et al, 1994) and a C-terminal carbohydrate-binding module (Xu et al, 1995) joined by a 20 residue proline-threonine linker with the sequence (PT)3T(PT)3T(PT)3 (Figure 1.4). When produced by C. fimi, the linker is O-glycpsylated with ot-mannosyl residues. The significance of this modification will be further discussed in Chapter 3. The family 10 Cex catalytic domain (CexCD) (Figure 1.5) is a 315-residue (ct/p)8-barrel retaining P-l,4-glycosidase belonging to the GH-A clan in the glycoside hydrolase superfamily of carbohydrate-active enzymes. The enzyme makes use of a glutamate (Glu233) as its catalytic nucleophile (Tull et al, 1991; Tull and Withers, 1994; White et al, 1996), as well as a second Chapter 1 - General Introduction 11 Figure 1.4. The modular architecture of Cex. Cex consists of an N-terminal 315 residue (a/B) 8-barrel catalytic domain (CexCD; PDB: 1EXO) (White et al, 1994) and a C-terminal 108 residue B-barrel cellulose-binding domain (CexCBD; PDB: 1EXG) (Xu et al, 1995), connected by a flexible proline-threonine linker (PT-linker). Also shown is the "beads-on-a-string" model of Cex, illustrating the catalytic and cellulose-binding domains tethered by a conformationally flexible PT-linker (drawn in an arbitrarily extend form), along with the structure of an a-D-mannopyranosylthreonine. The catalytic glutamic acid residues in the active site of CexCD and the exposed tryptophan sidechains of CexCBD forming the cellulose-binding surface are identified. Figure 1.5. X-ray crystal structure of the Cex catalytic domain. The ribbon representation of the X-ray crystal structure of the Cex catalytic domain (CexCD; PDB: 1EXO) (White et al, 1994). The catalytic nucleophile (Glu233) and the general acid/base residue (Glu 127) are highlighted in blue. Chapter 1 - General Introduction 13 glutamate (Glul27) as its catalytic general acid/base residue (MacLeod et al, 1994). Cex hydrolyzes xylan, and to a lesser extent, cellulose, endolytically (Charnock et al, 1998). Consistent with previous findings, detailed kinetic analysis performed on Cex using synthetic substrates showed that it has an ~ 80-fold preference for aryl p-xylobiosides over the aryl P-cellobiosides (Figure 1.6) (Gilkes et al, 1991a; Notenboom et al, 1998b). Using aryl glucoside substrates (Figure 1.6), the rate determining step (RDS) for CexCD is the initial glycosylation step of the double-displacement mechanism (Figure 1.1), in which the catalytic Glu233 nucleophilically attacks the anomeric centre of the substrate, thereby releasing the aglycone. This was determined by the strong dependences found in the Bronsted plots of the log(kcat) and log(kca t/Km) of CexCD against the leaving group abilities (pKa) of the phenols using a series of i aryl P-D-glucosides (Tull and Withers., 1994). It is important to note that kc a t reflects the rate of the slowest step in the enzyme mechanism, while kcat/Km reflects events up to and including the first irreversible step in the mechanism, which in this case is most likely the formation of the glycosyl-enzyme intermediate. In contrast, the deglycosylation step was determined to be rate-limiting when using cellobioside substrates, as evidenced by the lack of dependence in the Bronsted plots of the logfkcat) versus leaving group phenol pKa values (Tull and Withers, 1994). A range of pH-dependence studies on CexCD have been performed using the substrate 2,4-dinitrophenyl p-D-cellobioside (2,4-DNPC) to help ascertain key ionization events which are critical for enzymatic activities. The bell-shaped pH dependence ofk^/Km showed a basic ionization at pKa = 7.7 and a possible acidic ionization at pKa = 4.1 (Figure 1.7). This indicates that events leading up to the first irreversible step, the formation of the glycosyl-enzyme, most likely require two ionizable groups with the two indicated pKa values. From detailed kinetic studies using mutants of CexCD, it has been proposed that the decrease in kc a t / K m following an ionization at pKa = 7.7 can be attributed to the deprotonation of the general acid residue Glul27 (MacLeod et al, 1994), while the increase following an ionization at pKa = 4.1 can be assigned to the deprotonation of the catalytic nucleophilic Chapter 1 - General Introduction 14 Figure 1.6. Commonly used synthetic aryl substrates. Examples of an (top) aryl glucopyranoside, (middle) aryl B-xylobioside, and (bottom) aryl P-cellobioside are shown. Specifically, (top) 2,4-dinitrophenyl glucopyranoside, (middle) 2,4-dinitrophenyl P-xylobioside (2,4-DNPX2), and (bottom) 2.4-dinitrophenyl P-cellobioside (2,4-DNPC) are commonly-used substrates for studying Cex. Chapter 1 - General Introduction 15 16r 14 12 S 10 8 6 4 200 ^150 E 1« 100 3 5 0 Figure 1.7. pH-dependence of Cex towards 2,4-DNPC. The pH-dependence of (top) kcat and (bottom) kcat/Kn, of Cex using the substrate 2,4-DNPC (adapted from Tull and Withers, 1994). ^ a t / K m was fit to a bell-shaped activity profile with apparent pKa values of 4.1 and 7.7 (37 °C, with 50 mM citrate (pH 4-6), 50 mM phosphate (pH 6-8), and 50 mM Tris (pH 8-9)). Chapter 1 - General Introduction 16 residue Glu233 (MacLeod et al, 1996). This is consistent with the ionization states of these residues as required for the retaining double-displacement mechanism. Along similar lines of argument, the rate-limiting deglycosylation step might be expected to exhibit a pH-dependence for only one glutamate, namely the general base. Surprisingly, over a pH range of ~ 4 to 9 studied to date, kcat remains constant (Tull and Withers, 1994). This suggests that the pKa of the general base is below 4.0. Unfortunately, instability of the enzyme at lower pH conditions precluded measurements of this pKa value. Chapter 1 - General Introduction 17 1.3 CARBOHYDRATE-BINDING MODULES 1.3.1 Classification of carbohydrate-binding modules Carbohydrate-binding modules, or CBM's, were formerly referred to as cellulose-binding domains (CBD) based on the initial discovery of several modules that bind to cellulose (Gilkes et al, 1988; Tomme et al, 1988). Carbohydrate-binding modules are discretely folded regions within carbohydrate-active enzymes and have been classified, based on amino acid sequence similarities, into CaZy families 1 through 49 (www.CaZy.org) (Tomme et al, 1995b). CBM's and catalytic domains are often joined by flexible linker sequences in multi-domain enzymes. 1.3.2 CBM binding and polysaccharide hydrolysis Three general roles have been identified for CBM's to assist in substrate hydrolysis. These include: (1) a proximity effect, (2) a targeting function, and (3) a disruptive function (Boraston et al, 2004). Due to a linked CBM, catalytic modules can be concentrated on the polysaccharide substrate surfaces. The increased local concentration of the enzyme leads to the more rapid degradation of the polysaccharide substrate (Bolam et al, 1998). This phenomenon is exemplified by numerous studies in which the removal of the CBM's through proteolytic excision or genetic truncation led to significant decreases in the enzymatic activities on insoluble polysaccharides, but not soluble ones (Bolam et al, 1998; Boraston et al, 2003; Gilkes et al, 1988; Hall et al, 1995; Tomme et al, 1988). CBM's are also selective for the various types of major polysaccharides like cellulose, xylan, and mannan, and some can even target specific areas of a polysaccharide. For example, the family 9 CBM from xylanase 10A of Thermotoga maritima has specificity towards the reducing ends of polysaccharides, suggesting the possibility that it targets damaged regions of plant cell walls (Boraston et al, 2001; Notenboom et al, 2001). In addition, CBM's do not necessarily have the same specificity for the substrates that their attached catalytic domains hydrolyze. For example, Cex cleaves hemicellulose (xylan) preferentially, while its binding domain localizes the protein onto Chapter 1 - General Introduction 18 crystalline cellulose. Some CBM's also appear to be capable of disrupting polysaccharide structure, thereby enhancing the degradative capacity of the catalytic module. However, the generality of this phenomenon is uncertain, as this effect has only been observed in limited numbers of cases (Din et al, 1994; Gao et al, 2001; Gilkes et al, 1993). 1.3.2 The family 2 CBM from Cex The xylanase Cex from Cellulomonas fimi contains not only a family 10 catalytic domain, but also a family 2 CBM. For consistency with published literature, this will be referred to as the family 2 cellulose-binding domain (CexCBD). The physical linkage of the CexCBD to CexCD is critical for its hydrolytic activity towards natural substrates. Based upon its 3-dimensional structure, as solved by NMR spectroscopy (Xu et al, 1994), combined with detailed calorimetry studies (Nikolova, 1997), the mode of action of the CBD is mediated by three exposed tryptophans forming a hydrophobic binding surface to which "crystalline" matrices (in this case cellulose) bind. A more detailed discussion of the overall assembly of the catalytic and cellulose-binding domains in the intact Cex enzyme will be presented in Chapter 3. Chapter 1 - General Introduction 19 1.4 SPECIFIC AIMS OF THE THESIS The glycoside hydrolase family 10 xylanases are well characterized systems. The extensive structural, kinetic, and mutagenic studies on these enzymes provide a wealth of information describing the molecular features which underpin their ability to hydrolyze both xylo- and cello-oligosaccharide substrates with significant rate enhancements. As dynamic systems, the hydrolysis of these polysaccharides are often accompanied by subtle and/or dramatic conformational changes in both enzymes and substrates. The time dependencies of these changes are very difficult to characterize by structural-based biochemical approaches such as X-ray crystallography and small-angle X-ray scattering. Therefore, Chapter 2 will present the use of NMR spectroscopy to investigate the dynamic changes occurring along the catalytic pathway of the model system Cex. Specific focus is placed on comparisons between the apo-protein and its covalently trapped glycosyl-enzyme form (2FCb-CexCD). Chapter 3 will extend these NMR methods to characterize the dynamic glycosylated proline/threonine linker which tethers the catalytic domain of Cex to its cellulose-binding domain. Catalysis by Cex is primarily due to the interactions of ionizable groups within a complex hydrogen bonding network in its active site. Although kinetic studies can yield pH-dependence activity profiles for glycoside hydrolases, it is very difficult to interpret and translate this information into the specific charge states for ionizable groups. Through a variety of NMR-monitored titration studies, the electrostatic landscape of the Cex active site along its reaction pathway will be investigated in Chapter 4. These studies can help to define the charge states of the ionizable groups in both apo-CexCD and 2FCb-CexCD as they are poised to undergo the glycosylation and deglycosylation reactions, respectively. The majority of structural, kinetic, and dynamic studies on Cex were performed using synthetic mono- or di-saccharide-based substrates and inhibitors, whereas the natural substrates of the Chapter 1 - General Introduction 20 enzyme are extended polysaccharides. Chapter 5 will present the complementary use of X-ray crystallography and inactivation kinetics with two synthetic xylotriose inhibitors to probe for a possible glycone -3 binding subsite in Cex. These studies also yielded insights into'the aglycone +1 subsite of Cex, which to date has not been characterized structurally. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 21 Chapter 2 NMR Spectral Assignments of Cex and the Changes in Dynamics and Stability upon Formation of the Glycosyl-Enzyme Intermediate Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 22 2.1 INTRODUCTION Although significant progress has been made in understanding the structural basis for the catalysis provided by enzymes, often lacking are descriptions of the changes in thermodynamic and dynamic properties of these enzymes and their substrates that accompany binding and hydrolysis. The very nature of enzymes, with their fast turnover rates, essentially precludes the biophysical and biochemical study of reaction intermediates. Complexes with mechanistic-based inhibitors designed to mimic the intermediates and/or the transition states of these systems can often be "trapped" for a period of time sufficient for their study. The subsequent examination of structural stability gained or lost upon formation of enzyme intermediates can be insightful in dissecting the overall energy landscape of the reaction mechanism. The energetic studies of the transition of enzymes from a folded to an unfolded state are often performed using a variety of techniques including circular dichroism (CD) spectroscopy, calorimetry, hydrogen exchange by NMR spectroscopy, and partial proteolysis. However, the direct comparison of stabilities between the apo-enzyme and its intermediate is rare. Specifically, the available information for the comparison of glycoside hydrolases and their reaction intermediate is often anecdotal, involving general observations made regarding susceptibility to proteases (Connelly et al, 2000; Street et al, 1992). To investigate the site-specific changes in dynamics and thermodynamics along the reaction coordinate for glycoside hydrolases, the model xylanase Cex (C/XynlOA) from Cellulomonas fimi was studied. Specifically, the 315 residue catalytic domain in its free (apo-CexCD) and glycosyl-enzyme intermediate (2FCb-CexCD) forms were characterized. The latter species was trapped by the covalent modification of Glu233 with the mechanism-based inhibitor (or "slow-substrate") 2,4-dinitrophenyl 2-deoxy-2-fluoro-B-cellobioside (McCarter et al, 1993). Using NMR (relaxation, hydrogen exchange) and CD (thermal denaturation) spectroscopy, and combining with thermolysin proteolysis, it was demonstrated that the local and global stability of CexCD increases along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 23 timescale dynamics remain relatively unperturbed. Such behaviour, seen also with Bacillus circulans xylanase (Connelly et al, 2000), may reflect thermodynamically favourable interactions within the relatively rigid active site of CexCD necessary to bind, distort, and subsequently hydrolyze glycoside substrates. Chapter 2 - N M R Assignments, Dynamics, and Stability of Cex 24 2.2 MATERIALS AND METHODS 2.2.1 Protein expression and purification Full length Cex was expressed as described previously using pUC12-l.lCex(PTIS) in E. coli BL21(A,DE3) cells (O'Neill et al, 1986a). This plasmid contains the gene encoding Cex with an endogenous secretory sequence that is cleaved upon transport to the periplasm. The 70% 2 H and 99% 13C/15N-labelled (DCN-labelled) protein was produced starting with a 20 mL overnight culture in LB media (100% H 20) containing 100 pg/mL carbenicillin. The resultant cells were centrifuged, washed with unlabelled M9 media, and used to inoculate a 20 mL culture in M9 media prepared with 6 g Na2HP04, 3 g KH 2 P0 4 , 0.5 g NaCl, 120 mg MgS04, 11 mg CaCl2, 2.7 mg FeCl3, 100 mg carbenicillin, 1 mg thiamine, 3 g 99% 13C6-glucose, 1 g 99% 15NH4C1, and 1 g 99% 2 H/ 1 3 C/ 1 5 N-Celtone (Spectra Stable Isotope Inc.) in 700 mL of D 2 0 and 300 mL of H 2 0. After growth overnight, this culture was used to inoculate the remainder of the labelled M9 medium, which was then grown to an ODfsoo of ~ 0.5, induced with 0.1 mM IPTG, and harvested 48 hours later. The temperature was 30 °C throughout the entire growth and expression period. I5N-labelled Cex was also expressed using this protocol with 1 g 99% 15NH4C1 and l g of 99% 15N-Celtone (Spectra Stable Isotope Inc.) in 1 L of M9 media. The cells were harvested by centrifugation (6000 rpm, 20 min), resuspended in ~ 25 mL of 50 mM potassium phosphate, 0.02% NaN3 at pH 7.2 in H 2 0 (K-P-7 buffer), and lysed by passage twice through a French-press cell at 10,000 psi. After addition of 25 U (1 pL) of benzonase (Novagen) and 4 mg phenylmethyl sulfonyl fluoride, cell debris was removed by centrifugation (15000 rpm, 30 min), and the supernatant containing 2H/13C/15N-labelled Cex was loaded at a flow rate of 0.5 mL/min on a -150 mL FPLC column (GE Healthcare) packed with washed, autoclaved, and degassed long fibrous cellulose (Sigma) suspended in K-P-7 buffer. In the case of 15N-labelled Cex, cellulose was added directly to the pooled media supernatant and the lysed cells, stirred for 48 hours at 4 °C, and then Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 25 packed in an FPLC column. After washing the column with ~ 270 mL of 1 M NaCl in K-P-7 buffer, followed by ~ 200 mL of K-P-7 buffer, Cex was eluted with H 2 0 at 1 mL/min and collected in 10 mL fractions. The appropriate fractions were pooled and concentrated to ~ 3 mL using a 10 kDa MWCO polyethersulfone membrane (Pall Life Sciences) in a stirred ultrafiltration cell (Amicon Corp.), and buffer-exchanged to 100 mM Tris, 5 mM cysteine, and 2 mM EDTA at pH 8.0. Washed agarose-immobilized papain (150 pL) (Pierce) was then added to cleave Cex within the linker region, between its catalytic and carbohydrate-binding domains. After incubation overnight at 37 °C on a tube roller, the papain was removed by centrifugation at 5000 rpm. The agarose beads were rinsed three times with K-P-7 buffer, and all the supernatant fractions were combined and incubated overnight at 4 °C on a tube roller with 2 g of washed avicel (Fluka Biochemika). The avicel, with bound uncleaved Cex and the isolated carbohydrate-binding domain, was removed by centrifugation at 5000 rpm. The supernatant, containing CexCD, was concentrated by ultrafiltration using a 3 kDa MWCO membrane polyethersulfone (Pall Life Sciences) in a stirred ultrafiltration cell (Amicon Corp.), and exchanged into 20 mM potassium phosphate, 0.02% NaN 3 at pH 6.5 (NMR buffer). The final yields were 31 mg and 14 mg of 2H/ , 3C/1 5N-labelled and 15N-labelled CexCD, respectively, per liter of media. The enzyme concentration was determined spectrophotometrically using a predicted value of e280nm = 52870 M^cm"1 (ExPASy website http://ca.expasy.org/tools/protpar-ref.html) (Pace et al, 1995), and its purity was checked by ESI-MS and SDS-PAGE. 2FCb-CexCD was produced by incubating CexCD (~ 1 mM) with 2,4-dinitrophenyl 2-deoxy-2-fluoro-B-cellobioside (2F-DNPC) (McCarter et al, 1993) in a molar ratio of 1:3. The glycosyl-enzyme intermediate is stable on the order of many months as confirmed by kinetic reactivation studies (Tull and Withers, 1994), NMR spectroscopy, and mass spectrometry. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 26 2.2.2 NMR spectral assignment N M R spectra were acquired at 30 °C on a Varian kiova 600 M H z spectrometer equipped with a lW2B/"alsN pulsed field gradient probe. The backbone ' H N , 1 5 N , 1 3 C a , 1 3 C P and 1 3 C resonances o f 2 H / 1 3 C / 1 5 N - l a b e l l e d apo-CexCD (~ 1 m M protein in N M R buffer with ~ 10% D 2 0 as a lock solvent) were assigned.using the following suite o f 2H-decoupled sensitivity-enhanced TROSY-based 3-D triple-resonance experiments' with selection against ' H - , 3 C groups: H N C A , H N ( C O ) C A , H N C O , H N ( C A ) C O , H N ( C A ) C B , H N ( C O C A ) C B , N N - N O E S Y , and H N - N O E S Y (Yang and Kay , 1999). Due to the similarity o f its T R O S Y - H S Q C spectrum to that o f apo-CexCD, only the H N C A , H N ( C O ) C A , H N ( C A ) C B , and H N ( C O C A ) C B spectra were employed to assign the ' H N , 1 5 N , 1 3 C a , and 1 3 C P resonances o f 2 H / 1 3 C / 1 5 N - l a b e l l e d 2 F C b - C e x C D . Tryptophan ' H 6 ' and 1 5 N e I . signals were assigned from H N - N O E S Y and C 5 H E experiments (Yamazaki et al, 1993). Spectra were processed using N M R P i p e (Delaglio et al, 1995) and analyzed with S P A R K Y 3.0 (Goddard and Kneeler., 1999). ' H and 1 3 C shifts were referenced to external sodium 2,2-dimethyl-2-silapentane-5-sulfonate and 1 5 N shifts indirectly v ia gyromagnetic ratios (Wishart et al, 1995b). Reported chemical shifts were not corrected for an offset o f ( 'J N H ) /2 due to the T R O S Y selection, or for 2 H isotope effects. 2.2.3 Assignment validation by secondary structure prediction The secondary structure for folded proteins can be determined based solely on a comparison of the measured chemical shift differences ( 1 3 C a - 1 3 C p ) for each amino acid residue in the protein relative to the values expected for an unstructured polypeptide (Wishart and Sykes, 1994). From statistical and theoretical analysis, 1 3 C a chemical shifts move downfield or upfield for residues in a -helices or P-strands, respectively, relative to a random coi l state; the reverse holds true for 1 3 C P chemical shifts (Spera and Bax, 1991). Thus, the observed versus reference ( l 3 C a - , 3 C p ) shift differences provide a robust measure o f the secondary structure o f a protein. Note that this approach also approximately compensates for 2 H isotope effects on the 1 3 C a and 1 3 C P chemical shifts o f an Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 27 amino acid (Gardner and Kay, 1998; Venters et al, 1996). Fortunately, the X-ray crystal structures for CexCD (White et al, 1994) and 2FCb-CexCD (White et al, 1996) are available to act as references to compare with the structural predictions, with good agreement being indicative of the correctly assigned backbone nuclei by NMR spectroscopy. 2.2.4 Chemical shift perturbations to map structural changes Amide ' H N and 1 5 N chemical shifts are sensitive to their immediate chemical and electronic environments, and hence the binding of a covalent inhibitor will lead to chemical shift perturbations of amides undergoing structural changes. Based upon peak shifts in the 'H (A<%) and 1 5 N (Aa>N) dimensions, total residue-specific changes in frequency (A<y) can be determined using Equation 2.1: 2.2.5 1 5N relaxation measurements 'H - 1 5 N relaxation has been discussed in detail by others (Clore et al., 1990a; Clore et al., 1990b; Peng and Wagner, 1994), and hence a very brief overview is presented here according to Connelly et al. (Connelly et al, 2000). For a given peptide NH group, the three measured relaxation parameters (Ti, T 2, and the NOE) are functions of the spectral density, J(&), at that position in the protein. The spectral density depends on the amplitudes and frequencies of any internal motions which may be occurring at that site, as well as the global tumbling time (xm) of the molecule. J(co) is formally a function of five different frequencies: 0, coN, « H , {<*>H + *»N}, and {co H -03 N } . In the absence of any assumptions as to the form of /(oo), measurement of the three relaxation parameters T b T 2, and the NOE will always be insufficient to uniquely determine /(co). The isotropic "model-free" formalism of Lipari & Szabo (Lipari and Szabo, 1982a; Lipari and Szabo, 1982b) provides a suitable approximation for the spectral density, characterizing the internal motions of a particular 'H-1 5 N bond vector in terms of a generalized order parameter (S2) and an internal correlation'time (re), Equation 2.1. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 28 J((D) = S 2 x r ^ ^ ( 1 - S 2 )T^ 1 , 2 2 V1 + C0 T m j + V1 + C 0 2 T 2 j Equation 2.2. where S2 measures the degree of restriction of the motion and xe, contained in the relation 1/x = l/x m + l/x e, reflects the rate of these motions. The global xm (or for anisotropic tumbling, the diffusion tensor) is first calculated from a best fit of relaxation data for all well-ordered residues. S2 and xe are optimized for each site separately (Farrow et al, 1994). When xe « xm for a given peptide group, Equation 2.2 reduces to Equation 2.3 and only S2 and the global x m are needed to adequately represent molecular motion at that site. J ( c o ) = S 2 x r (1 + c o V ) . Equation 2.3. For some NH groups, conformational exchange on the ps to ms timescale (represented as Re X) can contribute significantly to the transverse relaxation process (T2), along with the usual dipole-dipole and chemical shift anisotropy terms ( R 2 D D and R 2 C S A , respectively): ^ - = R 2 D D + R 2 C S A + R E ^2 Equation 2.4. In other cases, where significant intramolecular motions occur at a particular NH site on two different time scales (one near xm), the extended model-free analysis developed by Clore et al. (Clore et al, 1990a; Clore et al, 1990b) can be applied to accurately fit the relaxation data. Here the original order parameter S2 is represented as a product of two order parameters, S f2 and S s 2 (with corresponding time constants Xf and xs) describing fast and slow internal motions, respectively. The equation for the spectra density then becomes Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 29 J(co) = S 2 T r < ( l - S f 2 ) ! , | ( 1 - S S 2 ) T 2 (l + C 0 2 T m 2 ) (l + < 0 2 T f ) (1 + C 0 2 T 2 2 ) Equation 2.5. where Xi and x 2 are defined by the relations l / i i = l / t m + l /x f and l /x 2 = l / x m + l/x s . 1 5 N relaxation measurements on 2H/ 1 3C/ 1 5N-labelled CexCD were carried out with a 600 M H z spectrometer at 30 °C using TROSY versions of T i , T 2 , and heteronuclear ' H - 1 5 N NOE experiments (Farrow et al, 1994). Steady-state 1 5 N heteronuclear N O E values were measured by recording spectra with and without 3 seconds of proton saturation and a total recycle delay of 5.016 seconds. Ti and T 2 values were obtained by nonlinear least-squares fitting of the cross-peak heights to the two-parameter equation for an exponential decay using the program CurveFit (Palmer, 1998). Heteronuclear N O E values were calculated as the ratio of the peak intensities recorded with and without proton saturation. Uncertainties in all spectra were estimated as described (Farrow et al, 1994) . Analysis of the 1 5 N relaxation data to obtain parameters describing the anisotropic global tumbling of CexCD, as well as internal dynamics according to the extended model free formalism of Lipari and Szabo (Kay et al, 1989; Lipari and Szabo., 1982a; Mandel et al, 1995), was carried out using the program TENSOR2 (Dosset et al, 2001). Data were fit to the above five models of internal mobility: S 2; S 2 and x e; S 2 and R^; S 2, x e, and R^; S 2, S f 2, and x s (Farrow et al, 1994; Mandel et al, 1995) . A chemical shift anisotropy term with a magnitude of 107 ppm was used for the tryptophan 1 5 N e l (Goldman, 1984; Hu et al, 1993). Amide and indole 1 5 N relaxation-dispersion data were acquired with relaxation-compensated ' H - 1 5 N CPMG-TROSY-HSQC experiments using a constant time C P M G - T 2 delay of 40 ms (Mulder et al, 2002). Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 30 2.2.6 Proton-deuterium exchange The exchange of labile protons in proteins with solvent depends upon their structural environment. In particular, intramolecular hydrogen bonding can lead to significant protection against HX. Thus, the exchange of these protected protons occurs only through high-energy structural fluctuations, as shown in the classic Scheme 2.1 (Englander et al, 1996): • ki k 2 Protein ( c l o s e d ) . . Protein ( o p e n ) * - Exchange k i Scheme 2.1. where k 2 is the "intrinsic" exchange rate of a given amide in a random coil polypeptide. The resultant observed rate of proton exchange can be defined as: k k k o b s = ' \ Equation 2.6. k_, + k 2 Most proteins exhibit proton exchange in the EX2 limit, where the refolding of the transient open state is fast compared with the intrinsic chemical exchange rate (k_i » k 2 ) , thereby simplifying Equation 2.6 into: k 0 b s _ vk-iy k 2 = K • k 2 Equation 2.7. where K is the equilibrium constant between the closed and open conformations of the protein. K, or its inverse termed the "protection factor," can be obtained from ko b s using calibrated k 2 values for a given set of experimental conditions. At high pH conditions or for marginally stable proteins, the chemical exchange can be more rapid than refolding (k 2 » k_i) . In this EX1 limit the observed exchange rate is simply the opening rate: k 0 b s = k i Equation 2.8. To measure the site-specific proton-deuteron exchange rates for Cex, samples of 15N-labelled apo- and 2FCb-CexCD (~ 0.4 mM) in 0.5 mL NMR buffer were lyophilized to dryness. Immediately after rehydration in an equivalent volume of D 20, a series of sensitivity-enhanced 'H- 1 5N TROSY-Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 31 HSQC spectra (Yang and Kay, 1999) were acquired over the course of 65 hours at 30 °C for each f sample. To detect the faster-exchanging residues, the first 6 spectra were recorded with 4 transients per FID (15 minutes total), while the latter spectra were recorded with 16 transients per FID for improved signal-to-noise. After scaling the spectra according to acquisition time, the exchange rates (kobs) were extracted from nonlinear least squares fittings of each peak's intensity versus time according to Equation 2.9 using SPARKY 3.0 (Goddard and Kneeler, 1999), where I(0) is the initial peak intensity, and \t) is the intensity after time t. I ( t ) = I ( 0 ) e ~ ( k ° b s ) t Equation 2.9. Data were also collected after 500 days of incubation at 4 °C in order to qualitatively compare the hydrogen-deuterium exchange behavior of the most slowly exchanging amides in apo-and 2FCb-CexCD. The latter sample contained a 5:1 molar excess of 2F-DNPC to help ensure complete inhibition over this time period. The pH* values after exchange were 6.5 for both samples. Control experiments using H 2 0 buffer demonstrated that CexCD retains its folded structure after rehydration. Predicted amide exchange rates, k2, for an unstructured polypeptide with the sequence of CexCD were calculated with the program Sphere (Zhang, 1995) using poly-D,L-alanine reference data corrected for amino acid type, pH, temperature, and isotope effects (Bai et al, 1993; Connelly et al, 1993). 2.2.7 Thermal denaturation measurements Thermal unfolding of apo- and 2FCb-CexCD were measured by CD spectroscopy using a 2 mm pathlength cell in a Jasco J-720 spectropolarimeter equipped with a Neslab RTE-111 heater and circulating water bath. The conformation of each protein sample (~ 0.20 mg/mL in 10 mM sodium phosphate, pH 6.5) was monitored at 220 nm at a heating rate of 1 °C/min. The midpoint unfolding temperature, T m , was obtained by non-linear least squares data fitting to a standard equation describing a two-state conformational equilibrium as given by Equation 2.10 (Santora and Bolen, Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 32 1992), where 0 f and 0° are the native and denatured baseline intercepts, m f and mu are the native and denatured baseline slopes, 9 is the observed CD signal, T is the temperature, AH is the enthalpy change, R is the gas constant (8.314 J K"1 mol"1), and T m is the temperature of the transition midpoint. 2.2.8 Thermolysin-catalyzed proteolysis Apo- and 2FCb-CexCD (0.50 mg/ml) were incubated at 50 °C with 0.20. mg/mL of thermolysin (Sigma) in 0.10 mM sodium acetate buffer (pH 5.5) containing 50 mM NaCl and 10 mM CaCl2 (Park and Marqusee, 2004). At designated time points, aliquots were withdrawn and the proteolytic reaction was quenched by adding EDTA to a final concentration of 15 mM. The quenched samples were analyzed using 15% SDS-PAGE gels, stained with Sypro Red fluorescent dye (Sigma), and scanned with a Typhoon 9400 imaging system (GE Healthcare). The intensities of the bands, corresponding to the intact protein, were quantified using ImageQuant software (GE Healthcare). T ) + ( 0 ° u + m u T (-AH/RT+AH7T m R) Equation 2.10. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 33 2.3 RESULTS 2.3.1 NMR spectral assignments The 34 kDa CexCD yielded excellent quality and well-dispersed NMR spectra, presumably due to its compact, stable (a/(3)8-barrel fold with a mix of secondary structural elements. Similar behaviour was seen with the (a/(3)8-barrel proteins tryptophan synthase (Vadrevu et al, 2003) and malate synthase G (Tugarinov et al, 2002). NMR spectral assignments for the dimeric triosephosphate isomerase from yeast have also been recently reported (Massi et al, 2006). The conventional and TROSY 'H- l 5 N HSQC spectra of CexCD, recorded with a 600 MHz spectrometer, were comparable in terms of resolution and signal-to-noise. However, TROSY-based triple resonance experiments, combined with aliphatic deuteration, were necessary to assign the signals from the mainchain nuclei of this protein via scalar correlations. These experiments, which involve magnetization both originating from and detected on the ' H N , require that amide deuterons, incorporated during biosynthesis in D 20, be exchanged for protons. This is usually accomplished via reversible unfolding-refolding in H20-based buffers (Gardner and Kay, 1998). Unfortunately, efforts to unfold CexCD using chemical (guanidinium hydrochloride and urea), pH, and thermal denaturation followed by an array of renaturing strategies failed to yield folded active protein. As a result, CexCD was expressed in 70% D 2 0 to allow for substantial deuteration, while ensuring a minimal 30% 'H occupancy of all amides. Additional D/H exchange occurred when the folded protein was purified and then stored in H20-based buffers for ~ 2 weeks at 4 °C prior to the commencement of NMR experiments. Nearly complete assignments of the resonances from the mainchain ' H N , 1 5 N, 1 3 C a and 1 3 C P nuclei in apo- and 2FCb-CexCD, as well as all 7 tryptophan indole 'H 6 l - I 5 N E l , were obtained using a suite of triple resonance experiments (Figure 2.1 and 2.2). Out of 304 non-proline backbone amides, 93% of the expected ' H N and 1 5 N signals in both apo- and 2FCb-CexCD were assigned. Similarly, Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 34 (aidd) N S I Figure 2.1. J H - 1 5 N TROSY-HSQC spectra of apo-CexCD. The 600 MHz 'H-^N TROSY-HSQC spectra of 2H/13C/15N-labelled apo-CexCD at 30 °C and pH 6.5 with backbone amide ' H ^ N and indole ' H 6 1 - 1 5 ^ 1 assignments indicated. Aliased signals are underlined, and the insets correspond to the central regions of the spectra, enlarged for clarity. Signals from arginine sidechains were not assigned, and those from the asparagine and glutamine 1 5 NH 2 groups were suppressed by this pulse sequence. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 35 Figure 2 .2. 'H-1 5N TROSY-HSQC spectra of 2 F C b - C e x C D . The 600 MHz 'H- 1 5N TROSY-HSQC spectra of 2H/13C/15N-labelled 2FCb-CexCD at 30 °C and pH 6.5 with backbone amide 'H N - , 5 N and indole 'H E l - I 5 N E l assignments indicated. Aliased signals are underlined, and the insets correspond to the central regions of the spectra, enlarged for clarity. Signals from arginine sidechains were not assigned, and those from the asparagine and glutamine 1 5 NH 2 groups were suppressed by this pulse sequence. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 36 94% of the 1 3 C a and 1 3 C P resonances in apo-CexCD and 93% in 2FCb-CexCD were identified. Missing assignments were possibly^due to spectral overlap, weak signals resulting from fast HX with water, and/or partial ' H N occupancy of slow-exchanging amides. A complete list of the chemical shifts of the assigned resonances for both apo- and 2FCb-CexCD are located in Appendix 1 and Appendix 2, respectively. The chemical shift assignments have also been deposited into the BioMagResBank (http://www.bmrb.wisc.edu/) under the accession numbers 7264 and 7265, respectively. 2.3.2 Secondary structure analysis The NMR-based secondary structure elements identified for apo- and 2FCb-CexCD are in good agreement with one another and with the (a/(3)8-barrel fold of both proteins determined by X-ray crystallography (due to the high degree of similarity, only the data for apo-CexCD is shown in Figure 2.3) (White et al., 1994; White et al., 1996). In addition, the 1 3 C P chemical shifts of the four Cys residues in CexCD are consistent with their involvement in disulphide bonds (Cysl67-Cysl99 and Cys261-Cys267) (Sharma and Rajarathnam, 2000). The confidence in the NMR assignments from this validation exercise allows for accurate structural interpretations of site-specific dynamics and stability data in the following sections. 2.3.3 Chemical shift perturbations upon glycosyl-enzyme intermediate formation To confirm the effects of covalent glycosyl-enzyme intermediate formation on the structure of CexCD, the ' H N and 1 5 N chemical shifts of corresponding residues in apo- and 2FCb-CexCD were compared. As summarized in Figure 2.3 and Figure 2.4, amide chemical shift perturbations were localized to residues within the vicinity of the active site. In contrast, the backbone nuclei in the remainder of the protein remained essentially unchanged upon covalent modification. This is consistent with the comparison of the X-ray crystallographic structures of apo- and 2FCb-CexCD Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 37 Figure 2.3. A summary of the HX, shift perturbation mapping, and secondary structure prediction. Row 1: The sequence of CexCD with the active site residues underlined (White et al., 1994; White et al., 1996). Row 2: Summary of the amide proton-deuterium HX kinetics of apo-CexCD, with open circles indicating ti/2 < 100 min, half-filled circles indicating 100 min < tm < 65 hrs, and filled circles indicating t]/2 > 65 hours at 30 °C; filled triangles identify amides protonated > 70% after 500 days of storage at 4 °C. Row 3: Amide *H N and15N chemical shift perturbations, due to the formation of the glycosyl-enzyme intermediate, are localized to the active site of the catalytic domain. Patterns of ( 1 3 C a - 1 3 C p ) chemical shift differences for apo- (Row 4) and 2FCb-CexCD (Row 5), relative to the values expected for a random coil polypeptide, are consistent with the secondary structure of CexCD determined by X-ray crystallography (Row 6 from PDB file 2EXO (White et al., 1994), with the oc-helices/B-strands have positive and negative ( 1 3C a- 1 3Cp) shift differences, respectively. Missing data correspond to prolines and residues with overlapped or unassigned NMR signals. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 38 Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 39 Figure 2.4. Chemical shift perturbations of CexCD upon covalent modification. The total chemical shift perturbations (Hz), as calculated using Equation 2.1 for all the available 'H- 1 5N amide resonances, are plotted in colour onto the ribbon diagram of CexCD. Green, blue, and red respectively represent shift perturbations of < 20 Hz, between 20 and 50 Hz, and > 50 Hz, while residues in gray are unassigned. The location of the 2F-cellobiosyl inhibitor is also shown. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 40 (PDB files 2EXO and 1EXP,'respectively), where only small conformational changes of the active site residues are present while an overall backbone rmsd of 0.16 A is maintained (White et al, 1994; White et al, 1996). Of these variations, the most notable is the outward displacement of the indole ring of Trp281 due to steric contacts with the hydroxymethyl group of the proximally-bound sugar (Notenboom et al., 1998b). This residue also exhibits the largest amide chemical shift difference (in Hertz) between the two forms of CexCD. Of the 7 tryptophan residues in CexCD, 3 (Trp84, Trp273, Trp281) are intricately involved in the binding of substrate and each of these show significant indole 'H^-^N 6 1 shift perturbations upon glycosyl-enzyme intermediate formation (Figure 2.1 and Figure 2.2). The lHel of the above mentioned Trp281 is hydrogen bonded to the carboxylate of Asp235, which in turn is bridged by His205 to the nucleophile Glu233. Trp84 stacks against the bound proximal sugar via ring interactions, with its H 6 1 hydrogen bonded to the general acid/base catalyst Glul27. Trp273 lies against the opposite face of the same sugar, while also donating a hydrogen bond to the C2-OH of the distal sugar. The formation of this hydrogen bond in 2FCb-CexCD likely leads to the pronounced downfield shift of- 1 ppm observed for the 'H 6 1 resonance of Trp273. 2.3.4 Backbone amide dynamics from 1 5 N relaxation measurements The backbone dynamic properties of CexCD were probed using 1 5 N relaxation measurements. Reliable T 1 ; T 2 , and heteronuclear NOE relaxation data were recorded for 199 and 194 of the possible 304 non-proline backbone amides in apo- and 2FCb-CexCD, respectively (Figure 2.5 and Figure 2.6; Appendix 3 and Appendix 4). The remaining residues were not analyzed due to spectral overlap and/or weak signals. The average Ti and T 2 values were 1.27 ± 0.04 s and 0.048 ±0 .0013 s, respectively for apo-CexCD, and 1.32 + 0.05 s and 0.047 + 0.0013 s for 2FCb-CexCD at 600 MHz and 30 °C. Fitting of the resultant T,/T 2 ratios with TENSOR2 (Dosset et al, 2001) yielded effective correlation times (xm) of 16.1 + 0.1 ns for the global tumbling of apo-CexCD and 16.8 ± 0.1 ns for Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 41 1.6-T T 1 1 1 1 1 r -0.07 0.05 H Residue Number Figure 2.5. 1 5 N T„ T 2 , NOE, and S2 for apo-CexCD. Plots of the measured 1 5 N T,, T 2 , and heteronuclear 'H-{I5N} NOE relaxation parameters and the fit anisotropic model-free order parameters S2 as a function of residue number for apo-CexCD. The secondary structural elements derived from the crystal structure of this protein (PDB entry 2EXO) are shown, with bars and arrows representing a-helices and B-strands, respectively. Missing data points correspond to prolines and residues with overlapped or unassigned NMR signals. The list of data values is located in Appendix 3. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 42 Residue Number § § P 55 Figure 2.6. 1 5 N T l 5 T 2 , NOE, and S2 for apo-CexCD. Plots of the measured 1 5 N Ti, T 2 , and heteronuclear 'H-{15N} NOE relaxation parameters and the fitted anisotropic model-free order parameters S2 as a function of residue number for 2FCb-CexCD. The secondary structural elements derived from the crystal structure of this protein (PDB entry 1EXP) are shown, with bars and arrows representing a-helices and P-strands, respectively. Missing data points correspond to prolines and residues with overlapped or unassigned NMR signals. The list of data values is located in Appendix 4. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 43 2FCb-CexCD. These measured values agree well with the expected t m of ~ 18.5 ns for a monomeric 315 residue globular protein at 30 °C (Daragan and Mayo, 1997). The 1 5 N T] and T 2 relaxation data were also fit to a fully anisotropic diffusion tensor using the crystallographically-determined structures of apo- and 2FCb-CexCD. For apo-CexCD, D z z = 1.18 (± 0.02) xlO7 s"1, D Y Y = 0.97 (± 0.02) xlO7 s"\ and Dxx = 0.94 (± 0.01) xlO7 s"1, whereas for 2FCb-CexCD, D z z = 1.11 (± 0.02) xlO7 s'1, D Y Y = 0.94 (± 0.02) xlO7 s1, and Dxx = 0.91 (± 0.01) xlO7 s"1. These values approximate the diffusion of an axially-symmetric prolate ellipsoid with D| \/D± of ~ 1.24 ± 0.02 for apo-CexCD and 1.19 ± 0.03 for 2FCb-CexCD. Furthermore, the principal axes of the fitted diffusion tensors are roughly co-linear with those of the corresponding moments of inertia calculated from the crystallographic co-ordinates of the two forms of CexCD, and the ratios of these experimental values agree with those predicted by HYDRONMR (apo-CexCD, observed 1.26 : 1.03 : 1.00, predicted 1.27 : 1.03 : 1.00; 2FCb-CexCD, observed 1.22 : 1.03 : 1.00, predicted 1.26 : 1.03 : 1.00) (de la Torre et al, 2000). Thus, the rotational diffusion behaviours of apo- and 2FCb-CexCD derived from 1 5 N relaxation measurements are consistent with the crystal structures of the prolate-shaped proteins. Based on these structures, HYDRONMR predicted a small increase of - 2% for the xm value of 2FCb-CexCD versus that of the apo protein. The slightly larger observed increase of - 4% may also reflect experimental errors and/or variations in exact sample conditions. The internal motional properties of the apo-CexCD and 2FCB-CexCD backbone amides on the ns-ps timescale can be extracted from the 1 5 N relaxation data by the extended anisotropic Lipari-Szabo model-free formalism in terms of a generalized order parameter, S2, which decreases from 1 to 0 with decreasing spatial restriction of the NH bond vector (Figure 2.5 and Figure 2.6; Appendix 3 and Appendix 4). Of the 199 amides analyzed in apo-CexCD, the relaxation behavior of 175 could be described using solely a S2 term (and hence xe for fast internal motions « xm), whereas 22 were best fit using both an S2 and xe term. Ala 18 fitted to S2 and an additional term, ReX, for conformational exchange, and only Glyl34 fitted to S2 and a second term, S f2 for motions on a timescale near xm. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 44 Furthermore, the average overall S2 value for apo-CexCD was 0.91 ± 0.04. Surprisingly, no evidence of enhanced flexibility on this timescale was detected even for the loop regions of the protein, as demonstrated by the average S2 values of 0.93 + 0.04, 0.90 ± 0.04, and 0.89 ± 0.05 for helices, strands, and loops respectively. Upon covalent modification to form 2FCb-CexCD, no noteworthy clianges in the fast timescale dynamics of the catalytic domain were detected. Of the 194 amides analyzed, the relaxation behavior of 174 fit solely to an S2 term, 19 to both a S2 and xe term, and only Gly97 required a S f2 term. Similar to the apo protein, 2FCb-CexCD exhibited an overall average S2 of 0.91 ± 0.05, with the helices, strands, and loops having average S2 values of 0.92 + 0.04, 0.92 + 0.04, and 0.89 ± 0.05 respectively. Most importantly, no significant changes occurred in the amide S2 values of the active site residues that could be measured in both forms of the protein. Together, these data indicate that CexCD has a rather rigid backbone with uniformly restricted sub-ns timescale mobility in both its unmodified and trapped glycosyl-enzyme intermediate forms. As is frequently the case (Boehr et al., 2006), we do not observe a correlation between the NMR-derived S2 order parameters and the X-ray crystallographic main chain thermal factors (B-values) of apo-CexCD, which are lowest for residues forming its (a/p)8-barrel core and increase approximately radially to become highest at the most exposed loop regions (2EXO.pdb) (White et al., 1994). Thus, these factors may represent crystal disorder or motions on timescales not detected by 1 5 N relaxation (i.e. not ns-ps or ms-ps). More importantly, the thermal factors do not change appreciably upon formation of 2FCb-CexCD (lEXP.pdb) (White et al., 1996), indicating that any such main chain motions or crystal disorder are also not influenced by the presence of the covalently-bound glycoside. To probe for ms-ps timescale backbone motions of CexCD, 1 5 N relaxation-dispersion measurements were also performed using a 600 MHz spectrometer. These experiments sensitively detect the contribution of conformational exchange to the effective decay of the transverse 1 5 N signal (Mulder et al, 2002). No evidence of such conformational exchange was observed for either apo- or 2FCb-CexCD (data not shown). Thus, the amide backbone of CexCD also appears uniformly rigid on Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 45 the ms-ps, as well as ns-ps, timescales, and formation of the glycosyl-enzyme intermediate does not measurably alter this behavior. 2.3.5 Tryptophan sidechain dynamics from 1 5 N relaxation measurements Similar tb the mainchain amides, the 7 tryptophan 1 5 N e l - ' H e l moieties of CexCD exhibited S2 values of 0.85 to 0.93 in both its apo- and glycosyl-enzyme intermediate forms (Appendix 3 and Appendix 4). Furthermore, no evidence of conformational exchange was observed for these groups in relaxation dispersion experiments. Thus, the indole sidechains of apo- and 2FCb-CexCD are also well ordered on the ms-ps and ns-ps timescales. 2.3.6 Amide and indole hydrogen exchange The proton-deuterium HX rate of an amide or indole depends on its structural and electrostatic environment, as well as local and global conformational fluctuations that allow contact with water. Most important are motions involving the disruption of hydrogen bonds (Englander et al, 1996). 'H- 1 5N HSQC spectra provide a convenient method for measuring the site-specific HX rates of a protein. Quantitative exchange rates, corresponding to 100 min < t1/2 < 65 hrs, were determined for 44 residues with well-resolved amide signals in apo-CexCD (Figure 2.3). Within this timeframe, exchange corresponds to local, rather than global, fluctuations of the protein (Englander et al, 1996). 56 residues with resolved signals exchanged too rapidly and the remainder too slowly over this time period for their respective HX rates to be determined accurately. In a similar fashion, HX measurements were carried out with 2FCb-CexCD. Quantitative exchange rates were determined for 31 amides in the inhibited protein with clearly resolved 'H- 1 5N HSQC signals while 66 residues with resolved signals exchanged too rapidly and the rest too slowly for accurate analysis. Note that the numbers of residues with rapid or with quantifiable exchange rates differed between apo- and 2FCb-CexCD in large part due to different patterns of 'H- 1 5N HSQC spectral overlap. As expected, the most rapidly exchanging amides in both forms of CexCD generally corresponded to residues on the Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 46 enzyme's surface, in exposed loops, at the N-termini of helices, or on the outside edges of P-strands, where they are readily accessible to the solvent and not involved in stable intramolecular hydrogen bonds. Insights into the effect of glycosyl-enzyme intermediate formation on the dynamic properties of CexCD can be obtained from comparison of the HX rates measured for corresponding residues in the unmodified and inhibited forms of this protein. Of the 21 amides for which quantitative exchange rates were measured in both apo- and 2FCb-CexCD, the average k^apo) / kobs(2FCb) =1.1 (Figure 2.7). Thus, the presence of the bound inhibitor does not significantly alter the most local, low energy fluctuations of CexCD that lead to HX over the relatively short timeframe of these measurements. However, it is notable that the active site residues Lys47 and His80 are clearly protected from HX upon glycosyl-enzyme intermediate formation, as evidenced by ko b S ( a p 0 ) / kobS(2FCb) ratios of > 50 and 25, respectively (Figure 2.8 A, B). These ratios are lower limits because the fast exchange rates of these amides in apo-CexCD could not be determined quantitatively. Similarly, of the 3 tryptophans in the active site of CexCD, the ! H E l of Trp281 exchanged too fast after transfer into D 2 0 buffer to be detected in any spectra recorded for either forms of the enzyme (tm < 15 min). Trp273, which donates a 'H E l hydrogen bond to the bound inhibitor, also exchanged rapidly in apo-CexCD, but was detectable in the initial spectra obtained with 2FCb-CexCD (15 min < t1/2 < 60 min). Trp84, which hydrogen bonds via its 'H E l to the general acid/base Glul27 (White et al., 1994; White et al., 1996), showed a 35 fold decrease in keX from 7 xlO"4 s"1 for apo- CexCD to 2.0 xlO - 5 s_1 for 2FCb-CexCD (Figure 2.8 C). The increased protection of the amides of Lys47 and His80 and the indole of Trp84 may reflect dampened local fluctuations or altered solvent accessibility or electrostatics due to the bound cellobioside. Further insights into the global dynamic properties of CexCD are provided by longer-timescale exchange studies. Quantitative HX measurements were carried out initially over a short time period to avoid any possible reactivation of 2FCb-CexCD. Somewhat surprisingly, after storage Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 47 2.5-r 2.0-2T O Residues Figure 2.7. Quantitative HX comparison between apo- and 2FCb-CexCD. The kobs(apo) / kobS(2FCb) for the 21 amide residues which have quantitative HX rates for both the free and glycosyl-enzyme bound states of CexCD are shown. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 48 ro <u Q. T3 <D N 75 E o 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 sSgSBl ° ° °° oo H80 Amide 2FCb apo 0 5,000 10,000 15.000 20.000 25,000 W84 Indole apo 22FCb " O 0 20.000 40,000 80,0001 100,000 200,000 300,000 Time (s) Figure 2.8. Qualitative comparison of HX between apo- and 2FCb-CexCD for selected active site residues. Formation of the glycosyl-enzyme intermediate stabilizes the active site of CexCD against local fluctuations leading to proton-deuterium HX. Shown are the normalized peak intensities versus time in D 2 0 buffer (pH* 6.5, 30 °C) for the amide 'H^s of Lys47 (A) and His80 (B) and the indole 'H E l of Trp84 (C) in apo- (open circles) and 2FCb-CexCD (filled circles). The insets are showing the same data, but expanded in the time axis. For the amides of Lys47 and His80 in 2FCb-CexCD and indole of Trp84 in apo-CexCD, exchange was either too slow or too fast to reliably fit a rate constant, and thus the lines are drawn only as a guide. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 49 for 500 days at 4 °C in the presence of excess 2F-DNPC, the protein remained fully inhibited as confirmed by its diagnostic 'H- 1 5 N HSQC spectrum. This permitted a qualitative comparison of the slow HX kinetics of CexCD in its unmodified and glycosyl-enzyme intermediate states. After this storage period, 40 and 61 resolved backbone amide residues remained visible in the 'H- 1 5N HSQC spectra of apo- and 2FCb-CexCD respectively, and had thus undergone less than ~ 30 % exchange (Figure 2.9 A, B). More significantly, as illustrated in Figure 2.9 C, the residues showing greater protection against HX in 2FCb-CexCD than in apo-CexCD clustered around the active site region of the protein. Thus, the presence of the bound intermediate also stabilizes the active site amides of CexCD against sub-global or global fluctuations that lead to HX. 2.3.7 Thermal denaturation To further investigate the effects of glycosyl-enzyme intermediate formation on the global energy landscape, the thermal stabilities of apo- and 2FCb-CexCD were monitored by CD spectroscopy (Figure 2.10). Since this enzyme does not refold reversibly under a wide range of conditions after thermal or chemical-induced denaturation, the data were fitted only to obtain midpoint unfolding temperatures. Although a reliable monitor of relative stability, this approach does not yield reversible thermodynamic parameters. Regardless, formation of the trapped glycosyl-enzyme intermediate significantly stabilizes the catalytic domain, as reflected by the measured T m values of 55.0 °C and 65.5 °C for apo-CexCD and 2FCb-CexCD, respectively. 2.3.8 Thermolysin-catalyzed proteolysis As rigorously demonstrated by Park and Marqusee (2004), proteolysis offers an alternative method for characterizing the local and global stability of proteins. Proteins in their folded conformations are generally resistant to proteolysis, unless they contain highly flexible polypeptide sequences in exposed loop or linker regions. Thus, to become proteolyzed, residues within structured Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 50 Figure 2.9. Covalent modification selectively protects active site amides from HX. Formation of the glycosyl-enzyme intermediate stabilizes amides surrounding the active site of CexCD against higher energy global or sub-global fluctuations leading to proton-deuterium HX. Shown are ribbon diagrams of (A) apo-CexCD and (B) 2FCb-CexCD with red and green balls, respectively, identifying the amides most highly protected from HX, i.e. undergoing < 30% exchange after storage in D 20 for 500 days at 4 °C and pH* 6.5. Only amides with unambiguously resolved HSQC signals are displayed. In panel (C), green balls denote residues showing < 30% exchange in 2FCb-CexCD and > 90% exchange in apo-CexCD (none exhibited the reverse pattern of slower exchange in the apo-protein). Only amides with unambiguously resolved signals in both forms of the protein are indicated. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 51 -14 -34 -i 1 1 1 1 1 27 37 47 57 67 77 Temperature (°C) Figure 2.10. CD-monitored thermal denaturation of apo- and 2FCb-CexCD. CexCD is stabilized against thermal denaturation upon formation of the glycosyl-enzyme intermediate as monitored using CD spectroscopy at pH 6.5. The fit T m values for apo-CexCD (solid line) and 2FCb-CexCD (dashed line) were 55.0 and 65.5 °C, respectively. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 52 regions of proteins must undergo conformational changes to higher energy states where cleavable sites are accessible to proteases. . We have used thermolysin-catalyzed proteolysis to compare the relative stabilities of apo-and 2FCb-CexCD. Under benign conditions, apo-CexCD is resistant to cleavage by this broadly specific, thermally-stable protease (as well as being resistant to papain, which is used to separate the catalytic and carbohydrate-binding modules of full length Cex). However, upon addition of sub-denaturing concentrations of urea (results not shown) or at elevated temperatures, the unmodified protein is readily degraded by thermolysin (ti/2 ~ 1 hr at 50 °C; Figure 2.11). Transient fragments of CexCD were not detected by SDS-PAGE, indicating that after a rate-limiting initial cleavage, the protein is destabilized to the extent that it is rapidly hydrolyzed to small polypeptides. Given that this temperature is approaching the T m value of apo-CexCD, it is likely that the initial cleavage occurs within the population of globally unfolded protein molecules. In striking contrast, 2FCb-CexCD was not measurably proteolyzed by thermolysin, even after 50 hours incubation at 50 °C (Figure 2.11). Thus, consistent with thermal denaturation studies, the native state of CexCD is markedly stabilized upon formation of the glycosyl-enzyme intermediate. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 53 1.0H "O ® 0.8 CO _CD O 0.6 c o 0 4 o C o. o.o H C O —Cr&Q- -Q : O- 0 • apo-CexCD O 2FCb-CexCD • » — • -0 1000 2000 3000 Time (min) Figure 2.11. Proteolytic degradation of apo- and 2FCb-CexCD by thermolysin. CexCD is stabilized against proteolytic degradation upon formation of the glycosyl-enzyme intermediate as monitored by thermolysin proteolysis of apo-CexCD (•, solid line) and 2FCb-CexCD (o, dashed line) at 50 °C and pH 5.5. The fractions of uncleaved protein were normalized to the fluorescence intensity of the uncleaved form of each protein, measured just prior to digestion. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 54 2.4 DISCUSSION In this study, we have used NMR and CD spectroscopy, along with thermolysin-catalyzed proteolysis, to characterize the catalytic domain of the B-l,4-glycosidase Cex and the consequences of its modification with a "slow substrate", 2,4-dinitrophenyl 2-deoxy-2-fluoro-B-cellobioside. This modification leads to a stable, yet catalytically competent, glycosyl-enzyme intermediate, thereby allowing a comparison of the structural, dynamic and thermodynamic properties of CexCD at two points along its double-displacement reaction pathway. 2.4.1 NMR spectral assignments and structural analyses As a requisite first step for the subsequent studies of Cex and CexCD using NMR spectroscopy, the 'H, 1 3 C , and 1 5 N resonances from the backbone amide and tryptophan indole nuclei in apo- and 2FCb-CexCD were assigned using TROSY-based NMR experiments, combined with 70% uniform sidechain deuteration. The a-helices and B-strands identified from chemical shifts agree closely with the crystallographically-determined structures of the catalytic domain. Furthermore, amide and indole chemical shift perturbations due to the covalent modification of CexCD are localized to residues within its active site region. Recognizing that chemical shifts are exquisitely sensitive to local conformational changes, this is consistent with the essentially identical crystallographic structures reported for CexCD in its apo-, covalent intermediate, and non-covalently inhibited states (Notenboom et al, 1998a; White et al, 1994; White et al, 1996). Together, these data indicate that CexCD adopts a very well-defined active site, poised for complementary interactions with a covalently-bound glycosyl-enzyme intermediate. Indeed, the defined conformation of its active site leads to the observed ~ 40 fold higher activity of Cex towards xylan relative to cellulose, as the sidechains of Gln87 and Trp281 must be displaced outwards to accommodate the hydroxymethyl groups present only in the latter substrate (Notenboom et al, 1998a). Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 55 2.4.2 Backbone amide and tryptophan indole dynamics Amide and indole 1 5 N relaxation and relaxation-dispersion measurements revealed that the backbone and tryptophan sidechains of apo-CexCD are uniformly rigid on the ns-ps and ms-ps timescales, respectively. Furthermore, as with.the mainchain crystallographic thermal factors of this domain, these dynamic properties did not change upon formation of the glycosyl-enzyme intermediate. In combination with, the above mentioned structural studies, this further supports the hypothesis that the active site of CexCD is a rigid scaffold, precisely positioning residues needed for the binding and subsequent hydrolysis of glycoside substrates. Interestingly, the primary amide of Gln87 and the indole of Trp281 displayed anomalously high thermal factors in the crystal structure of apo-CexCD (Notenboom et al, 1998a; White et al, 1994). Furthermore, the sidechain of Gln87 became unobservable in the crystal structure of 2FCb-CexCD due to unfavorable interactions with the hydroxymethyl of the distal sugar, yet well ordered in that of the 2-fluoroxylobiosyl-enzyme intermediate. Thus, some limited active site sidechain flexibility may be required for hydrolysis of both xylose- and glucose-based substrates by Cex. Indeed, the sidechains of Gln87 and Trp281 are displaced for cellobiosyl substrates due to the bulky nature of the additional hydroxymethyl groups present (Notenboom et al, 1998b; White et al, 1994; White et al, 1996). It is noteworthy that the (ct/p)8- or TDVI-barrel is one of the most common protein folds, with ligand or substrate-binding regions generally formed by loops grafted onto the core barrel (Nagano et al, 2002). Although it is not unexpected that amides within the ct-helices and P-strands are well ordered, it is somewhat surprising that the same behavior is exhibited by residues within the active site loop regions of CexCD. The (ct/p)8-barrel domain of malate synthase G also exhibits homogeneous backbone mobility in both its apo- and pyruvate/acetyl-CoA-bound states (Tugarinov et al, 2003; Tugarinov et al, 2004). However, 1 3 C and 2 H relaxation studies did reveal a dampening of methyl side chain dynamics in the active site of this enzyme upon substrate binding (Tugarinov et al, 2005). In agreement with molecular dynamics simulations, ms-us timescale backbone motions of an Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 56 active site loop, as well as 2 helices, in the dimeric triosephophate isomerase saturated with glycerol 3-phosphate were detected by recent NMR relaxation measurements (Massi et al, 2006). The generality of these observations will require backbone and sidechain dynamic studies of additional (a/p)8-barrel proteins. 2.4.3 Stabilization of CexCD by glycosyl-enzyme intermediate formation The dynamics and stability of CexCD were monitored by CD spectroscopy, thermolysin proteolysis, and HX studies. Unfortunately it was not possible to reversibly refold this protein, as required to measure the thermodynamic parameters governing its global folding/unfolding equilibrium. Nevertheless, as a qualitative indicator of stability, the T m value of CexCD increased by over 10 °C at pH 6.5 upon formation of the glycosyl-enzyme intermediate. Similarly, 2FCb-CexCD remained resistant to thermolysin under conditions where the apo enzyme was rapidly proteolyzed. Thus, as reported previously for several glycosidases, including Agrobacterium sp. P-glucosidase (Street et al, 1992) and Bacillus circulans xylanase (Connelly et al, 2000), the native structure of CexCD is dramatically stabilized in its glycosyl-enzyme intermediate state. By thermodynamic linkage, preferential non-covalent binding of a ligand to a folded protein must lead to net stabilization of the resulting complex relative to the unfolded protein and the free ligand. Indeed, irreversible deactivation studies combined with differential scanning calorimetry revealed that the apparent T m value of Cex increased by ~ 2 °C upon saturation with the weakly binding substrate, 4-nitrophenyl P-cellobioside (Nikolova et al, 1997). Such stabilization need not arise in the case of a glycosyl-enzyme intermediate, since the sugar remains covalently-bound to the enzyme in both its folded and unfolded states. However, inspection of the X-ray crystal structure of 2FCb-CexCD reveals an extensive network of hydrogen-bonding interactions between polar and ionic sidechains of the -2 and -1 subsites of the enzyme's active site with the hydroxyl groups of the bound fluorocellobioside, augmented by van der Waals contacts primarily involving aromatic residues, including the 3 previously mentioned tryptophans (Notenboom et al, 1998a; White et al, 1996). The former Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 57 enthalpically-favorable interactions (Tomme et al, 1996; Zolotnitsky et al, 2004) likely led to the observed stabilization of the glycosyl-enzyme intermediate of CexCD. Although less readily measurable and interpretable, entropic factors, including water release associated with the latter aromatic-sugar interactions, may also contribute (Zolotnitsky et al, 2004). Both qualitative and quantitative insights into the stability of CexCD are provided by NMR-monitored HX measurements. According to standard models of protein HX in the pH-dependent EX2 regime, the protection factor for a given amide, defined as the ratio of its predicted exchange rate in a random coil polypeptide, k2, to its measured value in a folded protein, k o b s , can be interpreted as the inverse of an equilibrium constant describing fluctuations between a closed, non-exchangeable state and a transiently-exposed, exchange-competent state. Thus protection factors provide a measure of the residue-specific free energy changes (AG°HX = RTln(k p r e d/ke X)) governing local or global conformational equilibria leading to exchange (Englander et al, 1996). Although not proven herein for CexCD, protein HX typically follows an EX2 mechanism under conditions such as those of this study, whereas the open-limited EX1 mechanism is usually observed only at elevated pH values or with destabilized systems (Englander et al, 1996). Based on these arguments, the protection factors for these slowly exchanging residues are > 107, and thus AG° of unfolding of apo-CexCD under native conditions is > 9 kcal/mol. Due to its elevated thermal stability, this free energy change must be even greater for 2FCb-CexCD. HX measurements also demonstrate the stabilization of CexCD upon formation of the glycosyl-enzyme intermediate. Although most weakly protected amides (t1/2 < 65 hrs) showed comparable exchange rates in the apo- and covalently-modified protein, it is notable that two active site residues, Lys47 and His80, in this category were > 25 and > 50 fold more protected in 2FCb-CexCD, respectively. Consistent with these effects, the labile sidechain amine H C 1 of K47 and imidazole ring H 8 1 of His80 are also protected from HX in 2FCb-CexCD (Poon et al, 2006; Schubert et al, 2007). Similarly, the indole of Trp84 was 35 fold more protected in the trapped glyeosyl-Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 58 enzyme intermediate than in the free enzyme. In the crystal structures of both forms of the enzyme, the amide of His80 is hydrogen bonded to a bound water molecule, the amide of Lys47 is weakly hydrogen bonded to the carbonyl oxygen of N44 within a reverse turn, and the indole of Trp84 is hydrogen bonded to the general acid/base Glul27. Given the lack of any clear structural perturbations or altered solvent accessibility for these residues, their increased HX protection in 2FCb-CexCD likely results from dampened local fluctuations (on timescales slower than those detectable by 1 5 N relaxation) due to the presence of the nearby bound inhibitor. Alternatively, changes in the electrostatic environment of the active site due to glycosylation, and hence neutralization, of the nucleophile Glu233, as well as the predicted deprotonation of the general acid Glul27 (Chapter 4; Mcintosh et al, 1996), could affect the rate of (OH) base-catalyzed HX. Even more pronounced differences between the two forms of CexCD are seen with the HX behavior of the most slowly exchanging amides. Although only qualitatively measured, 12 amides in apo-CexCD that exchanged extensively between 65 hrs and 500 days of storage in D 2 0 buffer remained well probated in 2FCb-CexCD. Note that this number corresponds to amides with unambiguously resolved HSQC signals in both forms of the protein, and is likely a lower limit of the number of residues actually showing greater HX protection due to the covalent modification of CexCD. As illustrated in Figure 2.9 C, these amides, which include Glu43, Aspl23, Asnl26, Asnl69, Trp273, and the nucleophile Glu233, cluster around the active site of the enzyme. Thus, formation of the glycosyl-enzyme intermediate also stabilizes this region of CexCD against higher energy sub-global or global fluctuations that lead to the slow HX of well-protected active site amide groups. A more detailed analysis of these fluctuations would require approaches such as "native state HX" measurements, in which exchange is measured as a function of increasing denaturant concentrations to progressively alter the free energy folding landscape of a protein (Bai, 2006). Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 59 2.4.4 Implications for catalysis Building upon previous enzymatic and X-ray crystallographic studies, the characterization of CexCD by NMR spectroscopy provides further insight into the catalytic mechanism of this model glycoside hydrolase. It is reasonable to assume that a marked stabilization, similar to that observed with 2FCb-CexCD, also results upon glycosyl-enzyme intermediate formation with natural substrates. The enzyme-substrate interactions responsible for this stabilization presumably facilitate catalysis by also lowering the energy of the transition state leading to this intermediate. Importantly, the covalent modification of CexCD does not measurably alter the already well-ordered backbone structure and restricted fast timescale mobility of this enzyme. A similar conclusion was reported for the family 11 B-l,4-xylanase from Bacillus circulans (Connelly et al., 2000). Such behavior is not) exclusive to these glycosyl hydrolases, as comprehensively reviewed by Boehr et al. (Boehr et al., 2006) and exemplified by a recent study showing that the backbone of the TEM-1 B-lactamase is also highly ordered in its apo-state (Savard and Gagne, 2006). In contrast, several enzymes do exhibit significant NMR-detectable dynamic changes along their reaction pathways that are often associated with rate-limiting conformational transitions (Boehr et al., 2006). The restricted backbone flexibility of these two glycosyl hydrolases may reflect the high stability of both enzymes, which, when secreted, are exposed to potentially harsh extracellular environments. Also, neither enzyme is subjected to allosteric regulation, and thus neither requires conformational flexibility to respond to an allosteric effector (Kern and Zuiderweg, 2003). Overall, these data support a hypothesis that the active sites of glycosyl hydrolases such as Cex are rigidly positioned in a predominantly "lock-in-key" manner to bind and subsequently hydrolyze their glycoside substrates. To date, high resolution crystallographic studies have been reported for CexCD in its apo-, non-covalently inhibited, and covalent glycosyl-enzyme states (Notenboom et al.; 1998a; Notenboom et al, 1998b; Notenboom et al, 2000; White et al, 1994; White et al, 1996) while lacking are descriptions of its Michaelis enzyme-substrate and -product Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 60 complexes. Nevertheless, based on detailed analyses of several related glycosyl hydrolases, it is likely that the substrate must be distorted for the aglycone to bind within the +1 subsite of CexCD (Davies et al, 199.8). Such substrate distortion from a chair to a postulated skew-boat conformation may facilitate direct, in-line nucleophilic attack by Glu233 at the anomeric center, with concomitant aglycone leaving group departure. Of course, some conformational mobility of CexCD must occur along the reaction coordinate, including substrate binding and distortion, oxocarbenium ion-like transition state formation, and product release. Yet this may require excursions to induced or pre-existing higher-energy states on timescales or amplitudes not sampled by the current study. For example, activated cellobiosides are hydrolyzed by CexCD with rate constants in the range of 102-103 s"1 for the glycosylation step and 10 s"1 for the rate-limiting deglycosylation step, which correspond to ps-s timescale events (Tull and Withers, 1994). Characterization of such transient conformational changes will require additional dynamic studies, including the investigation of methyl sidechain motions of Cex in its apo-, non-covalently inhibited, trapped glycosyl-enzyme intermediate, and product complexes by 2 H - and 1 3C-NMR relaxation methods and other complementary biophysical approaches. The spectral assignments of its catalytic domain open the door to such measurements, as well as to the study of active site electrostatic interactions, which are necessary to further understand the catalytic proficiency of this model glycosidase. Chapter 2 - NMR Assignments, Dynamics, and Stability of Cex 61 2.5 CONCLUSION In this Chapter, the ' H N , 1 5 N, 1 3 C a and 1 3 C P resonances for CexCD in its apo and covalently-bound forms were successfully assigned using a suite of 3-D triple-resonance TROSY-based NMR experiments. The subsequent secondary structure predictions obtained correlate well with the published crystal structures for the two forms of the enzyme, indicating that the NMR assignments are accurate. The resultant localized chemical shift perturbations with the formation of the trapped Cex intermediate are also consistent with previous crystallographic studies indicating that the structure of CexCD is not significantly altered upon intermediate formation. Most importantly, NMR relaxation experiments reveal that, on a ns-ps and ms-us timescale, the backbone of CexCD is rigid regardless of the presence of a covalently-bound inhibitor. While its local backbone dynamics do not significantly change, the global stability of the enzyme, as measured by HX, CD-monitored denaturation, and thermolysin proteolysis, is dramatically altered upon glycosyl-enzyme intermediate formation. This leads to the hypothesis that the increased stabilization is realized from the formation of a large number of hydrogen-bonding interactions between the enzyme active-site and the bound inhibitor. Using the NMR assignments, I will next examine the dynamics of full length Cex with a focus on its glycosylated proline-threonine linker and any potential interdomain interactions between CexCD and CexCBD when they are physically tethered (Chapter 3). Subsequently, I will investigate local electrostatic interactions in CexCD through site-specific pKa measurements (Chapter 4). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 62 Chapter 3 Flexibility of the Inter-Domain Linker in Cex Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 63 3.1 INTRODUCTION Cellulolytic organisms produce a battery of endo- and exoglucanases necessary for the hydrolysis of cellulose and hemicellulose. These glycoside hydrolases are typically modular, consisting of conserved catalytic and carbohydrate-binding domains (or modules), as well as possible ancillary domains, joined by variable linker sequences (Beguin et al, 1994; Gilbert et al., 1993; Gilkes et al, 1991b). In general, the constituent domains of glycoside hydrolases are structurally independent and exhibit some aspects of their respective functions when separated (Coutinho et al, 1993; Hall et al, 1995; Tomme et al, 1988). Thus, binding modules appear to facilitate catalysis by targeting and maintaining the proximity of the catalytic domains towards substrates within complex macromolecular systems, such as the plant cell wall, and by disrupting the structures of the polysaccharides within these systems (Boraston et al, 2004; Shoseyov et al, 2006). The synergistic activity of the catalytic and carbohydrate-binding domains in a glycoside hydrolase requires that they be covalently tethered to one another via a linker sequence of the appropriate length and/or flexibility. The functional importance of these interdomain linkers, which can range from only a few to over a hundred residues and are often rich in proline and hydroxyamino acids (Gilkes et al, 1991b), has been established largely through deletion studies (Black et al, 1996; Black et al, 1997; Srisodsuk et al, 1993). However, the physical properties of glycoside hydrolase linkers remain poorly defined. Early observations revealed that these sequences are often susceptible to proteolysis, leading to the hypothesis that they are exposed, flexible polypeptides joining independently folded catalytic and binding domains (Gilkes et al, 1988; Tomme et al, 1988; van Tilbeurgh et al, 1986). In support of this hypothesis, initial small-angle X-ray scattering (SAXS) studies on cellulases from Cellulomonas fimi and Trichoderma reesei indicated that these two-domain enzymes adopt an elongated tadpole-like shape with the head (catalytic domain) and tail (cellulose-binding domain) connected by relatively extended proline-rich and serine/threonine-rich linkers, respectively (Abuja et al, 1988a; Abuja et al, 1988b; Miller et al, 1995; Pilz et al, 1990; Shen et Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 64 al, 1991). This general view has been refined through more recent SAXS analyses demonstrating that native and chimeric cellulases from Humicola insolens and Pseudoalteromonas haloplanktis adopt an ensemble of tertiary structures with a distribution of interdomain separations that is attributable to the flexibility of the intervening serine/threonine-rich linker sequences (von Ossowski et al, 2005; Receveur et al, 2002; Violot et al, 2005). However, due to experimental limitations, the degree of flexibility of the linker within the native H. insolens enzyme could not be determined directly. A comparison of the dimensions measured for various cellulases by SAXS has also led to the suggestion that glycosylation favors-more extended conformations (von Ossowski et al, 2005; Receveur et al, 2002). This conclusion is complicated by the different linker sequences of these enzymes, particularly with respect to their proline-content. Regardless, such studies have led to a general model whereby some cellulases bind and cleave crystalline cellulose through a caterpillar-like motion mediated by a flexible interdomain linker (von Ossowski et al, 2005; Receveur et al, 2002). Alternatively, in the cases of glycoside hydrolases such as the endo/exocellulase E4 from Thermobifida fusca, the catalytic and cellulose-binding domains are effectively fused due to the absence of a linker sequence (Sakon et al, 1997). This may facilitate processivity by directing a bound cellulose strand into the active site of the enzyme. Linkers and/or catalytic domains can also play thermodynamically stabilizing roles towards the substrate binding domains, as exemplified by the resistence of the starch-binding domain against thermal and chemical denaturation (Williamson et al, 1992). Three-dimensional structures have also been determined by X-ray crystallography for two intact family 10 glycoside hydrolases with short linker sequences. Electron density was absent or weak for parts of the serine-proline rich linker of Streptomyces olivaceoviridis E-26 xylanase, while its catalytic and carbohydrate-binding domains were found to interact directly through a hydrophilic interface (Fujimoto et al, 2000; Fujimoto et al, 2002). Similarly, some of the linker residues were not observed between the non-interacting structured domains of Cellvibrio japonicus xylanase 10C (Pell et al, 2004a). By inference, these studies indicated that the linkers of such family 10 enzymes are crystallographically disordered, and hence flexible, but gave no direct measure of that flexibility. Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 65 To investigate, on a residue-specific basis, the modular structure and linker properties of a glycoside hydrolase in its unmodified and glycosylated states, NMR spectroscopy was used to characterize the xylanase Cex (or CfXynlOA) from Cellulomonas fimi. With the previously-mentioned modular assembly of a catalytic and a cellulose-binding domain spanned by a proline-threonine linker, Cex is not glycosylated when expressed in E. coli. When Cex is secreted by C. fimi, its PT-linker is O-glycosylated with ot-D-mannose and a-D-galactose, such that ~ 24 moles of hexose are found per mole of enzyme (Ong et al, 1994) (Figure 1.4). Early studies demonstrated that this modification led to the resistance of the Cex linker region against proteolytic degradation, but that glycosylation has no significant impact upon the kinetic parameters for hydrolysis of polymeric substrates (MacLeod et al, 1992). Without a method for the overexpression of Cex in C. fimi, only very small quantities of the endogenously glycosylated protein can be obtained. Fortunately, using Streptomyces lividans as an expression host, Cex can be expressed and glycosylated in milligram amounts, as required for NMR spectroscopic characterization (MacLeod et al, 1992; Ong et al, 1994). Glycosylated Cex from S. lividans is reported to contain -17 moles of mannose, with traces of galactose, per mole of enzyme (MacLeod et al.) 1992; Ong et al, 1994). These differences are minor as samples of O-glycosylated Cex from C. fimi and S. lividans have very similar kinetic properties and are both resistant towards proteolytic degradation to the same extent (Langsford et al, 1987; MacLeod et al, 1992). Based on chemical shift, 1 5 N relaxation, and amide residual dipolar coupling (RDC) measurements, it was demonstrated that the catalytic and cellulose-binding domains are physically independent and joined by a PT-linker that is conformationally dynamic on the ns-ps timescale. Glycosylation does not perturb either domain, yet partially dampens the mobility of the linker. These data complement previous proteolytic, crystallographic, and SAXS studies, confirming directly the hypothesis that the PT-linker is a flexible tether, joining the catalytic domain and cellulose-binding modules of Cex as two "beads-on-a-string". Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 66 3.2 MATERIALS AND METHODS 3.2.1 Expression and purification of uniformly 15N-labelled Cex, CexCD, andCexCBD Non-glycosylated 15N-labelled Cex, encoded by the plasmid pUC 12-l.lCex(PTIS) (O'Neill et al, 1986b) was expressed using E. coli BL21 (^DE3) cells grown in M9 media enriched with 1.0 g/L 15NH4C1 (Spectra Stable Isotopes Inc.). 15N-labelled Cex, and the papain-cleaved product CexCD (residues 1-315), were expressed and purified by cellulose affinity chromatography, as described earlier (Chapter 2; MacLeod et al, 1994; Poon et al, 2007a). 15N-labelled CexCBD (residues 336-443) was expressed directly using the plasmid pTug-KH6-IEGR-CBM2a in E. coli BL21 (A.DE3) cells (Graham et al, 1995; McLean et al, 2000) utilizing M9 media. The N-terminal His6-tag was not removed after purification with Ni+2-affinity chromatography. The resulting proteins were > 95% pure as determined by SDS-PAGE and ESI-MS (predicted masses for unlabelled Cex, 47192 Da; CexCD, 34230 Da; His6-CexCBD, 12272 Da). Protein concentrations were determined spectrophotometrically using predicted e280nm values of 81440 M^cm"1, 52870 M^cm"1, and 28570 M" 'cm'1 for Cex, CexCD, and CexCBD, respectively (ExPASy website: http://ca.expasy.org/tools/protpar-ref.html).-3.2.2 Expression of 1 5 N Thr-labelled Cex Non-glycosylated 15N-Thr-labelled Cex was produced by expression of the plasmid pUC12.1.1Cex(PTIS) in E. coli BL21 (^DE3) cells grown in a synthetic medium (Muchmore et al, 1989) enriched with 200 mg/L of L-threonine (15N, >98%) (Cambridge Isotope Laboratory). 3.2.3 Expression of glycosylated 1 5 N Thr-labelled Cex Glycosylated 15N-Thr-labelled Cex was produced using the plasmid pIJ680-cex (MacLeod et al, 1992) in S. lividans 66 (TK64) cells. Transformed S. lividans stock (50 uL), stored in DMSO at -Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 67 80 °C, was used to inoculate three 5 mL tryptic-soy broth cultures (EM Science), containing 20 ug/mL of thiostrepton (Sigma), in 50 mL sterile Falcon tubes. After growth in an incubator shaker at 30 °C for 48 hours, the cells were collected by centrifugation and each tube was used to inoculate 1.5 L of a synthetic medium (Muchmore et al, 1989), containing 20 ug/mL thiostrepton, 0.016% antifoam C (Sigma), and 300 mg L-threonine (15N, >98%), in a 3 L beveled flask. The three cultures (totaling 4.5 L) were grown in an incubator shaker at 30 °C for 4 days, after which the S. lividans mycelium was removed by vacuum filtration through a Whatman glass microfibre filter. The supernatant, containing secreted glycosylated Cex, was incubated with CF-1 cellulose and purified by affinity chromatography, as described previously (Chapter 2; MacLeod et al, 1994; Poon et al, 2007a), yielding 37 mg of protein from 4.5 L of isotopically-enriched media. The purified protein was found to be heterogeneously glycosylated by SDS-PAGE and MALDI-TOF MS analyses. 3.2.4 NMR spectroscopy NMR spectra were acquired at 30 °C on a Varian Inova 600 MHz spectrometer equipped with a gradient triple resonance cryogenic probe. All proteins (~ 0.1 to ~ 0.4 mM) were in 20 mM potassium phosphate, 0.02% sodium azide at pH 6.5 with ~ 10% D 2 0 added as a lock solvent. Spectra were processed using NMRpipe (Delaglio et al, 1995) and analyzed with SPARKY 3.0 (Goddard and Kneeler, 1999). The mainchain ' H N , 1 3 C , and 1 5 N assignments of CexCD were previously determined through a suite of TROSY-based experiments, as described previously (Chapter 2; Appendix 1; Poon et al, 2007a). The *H N and 1 5 N assignments of CexCBD were based on those reported previously (Xu et al, 1995). CexCBD weakly self-associates as revealed by concentration-dependent amide or indole chemical shift perturbations for several residues, including Trp350, Trp387, and Trp405 of the proposed cellulose-binding surface (Xu et al, 1995). Thus, 'H- 1 5N HSQC spectra were recorded for a dilution series of CexCBD samples (~ 0.1 to ~ 0.4 mM) in order to obtain concentration-independent amide chemical shifts. Chapter 3 - Flexibility of the InterTDomain Linker in Cex 68 The calculation of the chemical shift differences between corresponding amides in the respective isolated catalytic and cellulose-binding domains and those in the full length protein can be performed to probe the potential for domain interactions when physically tethered. Likewise, the comparison of the same chemical shifts with those from glycosylated Cex can yield information about the, role of glycosylation in any potential domain interactions. The quantitative shift perturbations were calculated using Equation 2.1, described in Chapter 2. ' r j N - ^ N RDC values, 'DNH, were measured from 600 MHz sensitivity-enhanced HSQC spectra of 15N-labelled Cex (~ 0.5 mM, pH 6.5, 30 °C) in which the 1 5 N signal was recorded in the TROSY mode, whereas that of the ' H N signal was recorded in either the TROSY or anti-TROSY mode (L. E. Kay, personal communication to L. P. Mcintosh). After acquiring a control spectrum, partial protein alignment was achieved by the addition of Pf 1 bacteriophage (Profos AG, Regensburg, Germany) to a concentration that produced 13 Hz splitting in the 2H-NMR signal of the lock *H02H (Hansen et al, 2000). The 'DNH values were obtained from the differences between the corresponding TROSY and anti-TROSY *H N chemical shifts in the aligned versus control spectra of Cex. The axial (Da) and rhombic (R) components of the alignment tensors for the residues in catalytic and cellulose-binding domains were estimated using a Matlab routine for fitting 'DNH distribution histograms (Clore et al, 1998), as previously described (Skrynnikov et al, 2000). 1 5 N relaxation measurements were performed using TROSY-based Tu T 2 , and heteronuclear 'H-f^N} NOE experiments (Farrow et al, 1994; Yang and Kay, 1999). Steady-state heteronuclear 'H-I^N} NOE values were measured by recording spectra with and without 3 seconds of proton saturation and a total recycle delay of 5.016 seconds. Analysis of the 1 5 N relaxation data was carried out using the programs SPARKY 3.0 (Goddard and Kneeler, 1999) and TENSOR2 (Dosset et al, 2001). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 69 3.3 RESULTS 3.3.1 Non-glycosylated Cex expressed from E. coli 3.3.1.1 Structural independence between the catalytic and cellulose-binding domains The 'H- 1 5N TROSY-HSQC spectra of the isolated CexCD and CexCBD are shown in Figure 3.1 A and Figure 3.1 B, respectively. These spectra form the basis of the spectrum of the full length non-glycosylated Cex in Figure 3.1 C. The remarkably well-dispersed spectrum of this 443 residue (~ 47 kDa) protein overlaps almost exactly with the sum of the spectra of the individual modular domains. Assuming that peaks with the closest ] H N and 1 5 N chemical shifts in the overlapped spectra of the three species correspond to the same residue, the amide resonances from full length Cex were comparatively assigned based on the reported assignments of its constituent domains (Chapter 2; Appendixl; Poon et al, 2007a; Xu et al, 1995). The remaining signals in the TROSY-HSQC spectrum of Cex, all of which fall within the crowded region of - 7.9r8.4 ppm in the 'H dimension, were attributed to linker threonines, as discussed below. The ' H N and 1 5 N chemical shifts of an amide are strongly dependent upon its environment within a protein, and thus shift perturbations provide a very sensitive measure of potential structural interactions. The lack of any significant chemical shift differences, as shown from Figure 3.2, strongly indicates that the two modules of Cex do not non-covalently interact when tethered by the PT-linker. To confirm this conclusion, 'H-^N TROSY-HSQC spectra of full length Cex were also recorded under identical conditions before and after treatment with papain to cleave the PT-linker in situ. Without any exceptions, the resolved resonances observed from residues of the catalytic and cellulose-binding modules retained the same chemical shifts before and after proteolysis of the linker (Figure 3.3). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 70 105 110 115 120 125 130 B 12 11 10 10 0 0 o i f ^ * % ft. % 12 11 10 9 8 1 H (ppm) i — 6 105 110 115 120 125 130 100 105 I a. 110 115 120 125 130 Figure 3.1. ! H - , 5 N TROSY-HSQC spectra for CexCD, CexCBD, and Cex. The 'H- 1 5N TROSY-HSQC spectra of uniformly 15N-labelled (A) ~ 0.4 mM CexCD, (B) ~ 0.1 mM CexCBD, and (C) ~ 0.4 mM non-glycosylated Cex, recorded at 30 °C and pH 6.5. Signals from backbone amides and indole sidechains in the (red) catalytic domain, (blue) cellulose-binding domain, and (green) PT-linker were coloured manually to allow a visual comparison of the spectra. The amide H - N assignments of Cex (not labelled for clarity) were based on those reported for the isolated CexCD (Poon et al, 2007a) and CexCBD (Xu et al, 1995). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 71 14-12-10-1 315 336 443 CexCD CexCBD Residue Number Figure 3.2. Amide chemical shift perturbation between non-glycosylated Cex and the isolated CexCD and CexCBD. The calculated amide chemical shift perturbation (in Hertz) between the ^ re-labelled full length non-glycosylated Cex (from E. coli) and the isolated 15N-labelled CexCD and CexCBD is shown. The lack of significant shift perturbations indicate that the domains, when physically tethered by the PT-linker, remain independent and do not interact non-covalently. Data corresponding to prolines and overlapped and/or unassigned peaks are not plotted. Note that the typical 'H line width of an amide signal in these spectra is ~ 35 Hz with data processed to 5 Hz/point. For comparison, formation of a trapped glycosyl-enzyme intermediate leads to amide chemical shift perturbations in excess of 100 Hz (Figure 2.3). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 72 443 CexCD Residue Number CexCBD Figure 3.3. Amide chemical shift perturbation between non-glycosylated Cex and the in situ separated CexCD and CexCBD. The calculated amide chemical shift perturbation (in Hertz) between the 15N-labelled full length non-glycosylated Cex (from E. coli) and the in situ separated ^ r e -labelled CexCD and CexCBD is shown. The lack of significant shift perturbations confirm that the Cex domains, when together in solution, are behaving independently regardless of being physically tethered or not. Data corresponding to prolines and overlapped and/or unassigned peaks are not plotted. Note that the typical ! H line width of an amide signal in these spectra is ~ 35 Hz with data processed to 5 Hz/point. For comparison, formation of a trapped glycosyl-enzyme intermediate leads to amide chemical shift perturbations in excess of 100 Hz (Figure 2.3). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 73 3.3.1.2 Interdomain mobility between the catalytic and cellulose-binding domains The global dynamic properties of intact Cex were compared to those of CexCD and CexCBD using 1 5 N relaxation measurements. Based on amide 1 5 N Ti and T 2 values, the effective correlation times (tm) for the tumbling of the catalytic and cellulose-binding domains in full length Cex (0.4 mM) were determined to be 17.6 ± 1 . 1 and 14.3 ± 1.0 ns, respectively. As expected due to their physical linkage, these values are, respectively, higher than those measured for the isolated 34 kDa CexCD (16.1 ± 0.1 ns; ~ 0.4 mM) and 12 kDa CexCBD (8.4 ± 0.1 ns; ~ 0.1 mM). Also, whereas the anisotropic diffusion tensor of CexCD (with D z z : Dyy : D „ equal to 1.26 : 1.03 : 1.00) is consistent with its crystallographically-determined prolate ellipsoid shape (Chapter 2; Poon et al, 2007a), that of the corresponding catalytic domain in Cex (2.1 : 1.6 : 1.0) is distinctly anisotropic due to the attached linker and cellulose-binding domain. Similarly, the diffusion tensor of the cellulose-binding domain of Cex differs from that of CexCBD due to the attached linker and catalytic domain. Most importantly, however, the observation that the effective xm values of the two domains within Cex differ significantly from one another and from that of ~ 25 ns predicted for a globular 47 kDa protein (Daragan et al, 1997) indicates that they tumble semi-independently while tethered to one another by the PT-linker. Additional evidence for the structural and dynamic independence of the catalytic and cellulose-binding domains within native Cex is provided by amide ' D N H RDC measurements. RDC's, which arise as a result of the weak alignment of a macromolecule in a magnetic field, provide information on long-range orientations (i.e. the relative orientations of two protein domains to the external magnetic field). In the case of a multi-domain protein with minimal interdomain mobility, the average orientation of each domain will be described by a single, common molecular alignment tensor. In contrast, if medium- to large-scale interdomain motions occur, then the average orientations of the domains may differ due to different weak interactions with the alignment media, thereby leading to distinct alignment tensors (Braddock et al, 2001). Using TROSY-based HSQC Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 74 experiments, 'DNH values were measured for 151 and 54 amides belonging to the catalytic and cellulose-binding domains, respectively, in full length Cex. Inspection of the histograms showing the resulting ' D N H RDC distributions (Figure 3.4) reveals that the two domains indeed have distinct alignment tensors. This conclusion is confirmed quantitatively by fitting the histograms (Skrynnikov et al, 2000) to yield values of -10 Hz and 0.6 for the axial (Da) and rhombic (R) components of the alignment tensor for the catalytic domain, respectively, as compared to +9 Hz and 0.4 for the cellulose-binding domain. Thus, the two domains within Cex are oriented at least semi-independently by differential steric and electrostatic interactions as observed with Pfl phage (Zweckstetter et al, 2000), rather than behaving as a single rigid body. 3.3.1.3 Conformation ally dynamic and unstructured PT-Linker In addition to signals from its catalytic domain and cellulose-binding module, the 'H- 1 5N TROSY-HSQC spectrum of uniformly 15N-labelled Cex contains several peaks, clustered near 7.9-8.4 ppm in the ! H dimension, that likely arise from the threonine amides within the PT-linker. The 'H- 1 5N TROSY-HSQC spectrum of Cex, selectively labelled with 15N-Thr, was recorded to help resolve and identify these signals (Figure 3.5 A). This simplified spectrum contains a set of well-dispersed signals, combined with a group of sharp, overlapping peaks. Based on the reported assignments of CexCD and CexCBD, the former signals can be readily assigned to threonines within the structured catalytic and cellulose-binding domains. Thus, the remaining identifiable peaks must arise from the 11 threonines in the PT-linker. Due to their spectral overlap, as well as the repetitive nature of the .linker sequence, it was not possible to assign these signals to specific residues without additional approaches, such as the preparation of an expensive l 3C/ 1 5N-Pro and -Thr labelled protein sample (Kanelis et al, 2000). Nevertheless, it is readily apparent that all of the linker threonines have poorly dispersed amide *H N chemical shifts within the range expected for a random coil polypeptide (a distribution of ~ 4 ppm in 1 5 N chemical shift is expected due to nearest neighbor threonine and proline effects) (Wishart et al, 1995a). This indicates that, either the PT-linker lacks any predominant Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 75 201 1 5 10 12n B 1 1 c o 2H I 3H 1 II m t n m m m m i n i n m m m m m L O i n m N M T - ^ r- r- t 1 I + + + <r- V - -r-'DNH(Hz) Figure 3.4. Amide 'D N H RDC measurements for the catalytic domain, cellulose-binding domain, and the PT-linker region of non-glycosylated Cex. Interdomain mobility in Cex is confirmed by ' D N H R D C ' S measured for amides in the (A) catalytic domain, (B) cellulose-binding domain, and (C) PT-linker region, of the full length enzyme. The different maximum, minimum and most frequent values of these distributions reveal that the two domains and the PT-linker have distinct alignment tensors describing their different average orientations with respect to the magnetic field due to weak interactions with Pf 1 phage (Braddock et al., 2001; Clore et al., 1998). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 76 T 5 1 M05 T 4 3 1 T 4 2 4 T 1 6 3 T 1 0 9 T 3 9 0 T 2 7 6 T 3 T-L-m mo T 4 4 1 ' T - U I - 6 . j T ^ . T _ M15 T374 1355 , T 3 6 3 T-L-t T-L-u &T4sO T392 Q 1 2 4 9 M20 T438 . : T359 T 3 9 9 T 2 7 1 M25 8.5 8.0 7.5 1H (ppm) 9.5 9.0 8.5 8.0 7.5 'H (ppm) 7.0 6.5 Figure 3.5. 'H-^N TROSY-HSQC spectra of 15N-Thr-labelled non-glycosylated and glycosylated Cex. The 'H-^N TROSY-HSQC spectra of selectively ,5N-Thr-labelled Cex in its (A) non-glycosylated and (B) glycosylated forms reveal that the PT-linker is unstructured and that glycosylation does not induce any predominant structure. The well-dispersed signals are assigned to threonine amides within the (red) catalytic and (blue) cellulose-binding domains, based on comparisons to the spectra of CexCD and CexCBD. The remaining peaks, with random coil ' H N chemical shifts, correspond to threonine residues in the non-glycosylated and glycosylated PT-linker. These peaks were not specifically assigned and are identified alphabetically with the prefix T-L. Additional weak signals are attributed to (A, B) cis-trans isomerization of the Thr-Pro amides, along with (B) heterogeneous glycosylation. Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 77 structure, or that the repetitive linker sequence led to a repetitive structure, which does not result in the dispersion of the threonine amide chemical shifts. Based on relaxation measurements, as discussed below, the former conclusion is strongly favored. In addition, close inspection of the 'H- 1 5N TROSY-HSQC spectrum of 15N-Thr-labelled Cex reveals the presence of several weak peaks within this narrow chemical shift range. These most likely reflect conformational heterogeneity of the unstructured linker due to cis-trans isomerization of the Thr-Pro peptide bonds. To directly probe the dynamic properties of the non-glycosylated PT-linker, steady-state heteronuclear 'H-{1SN} NOE measurements were also carried out with selectively l 5N-Thr labelled Cex. The heteronuclear NOE is a sensitive indicator of sub-ns timescale dynamics, with decreasing values corresponding to increasing mobility of the amide 'H- 1 5 N bond vector (Kay et al, 1989). As shown in Figure 3.6 A, threonine amides within the catalytic and cellulose-binding domains have uniformly high heteronuclear NOE values, consistent with their well-structured environments. In marked contrast, those from the PT-linker exhibit low or negative heteronuclear NOE values, clearly demonstrating that they are conformationally flexible on the ns-ps timescale. As a further reflection of this flexibility, 5 linker threonines with resolved signals in the 1 5 N Ti, T 2 , and heteronuclear NOE relaxation spectra recorded for uniformly 15N-labelled Cex, were fit to obtain an average value of ~ 0.45 for the model-free order parameter, S2 (and an effective x c of ~ 6 ns). This is in comparison to the amides in the catalytic and cellulose-binding domains which displayed average S2 values of - 0.9 (Chapter 2) (Lipari and Szabo, 1982a; Poon et al, 2007a). This generalized Lipari-Szabo order parameter decreases from 1 to 0 with decreasing spatial restriction of the NH bond vector. It is also noteworthy that PT-linker threonines with resolved signals in the spectrum of uniformly 15N-labelled Cex yielded relatively small amide ' D N H RDC values ranging from -8 to 0 Hz (Figure 3.4 C). Although this could be due to well-aligned 'H-^N bond vectors at angles with respect to the magnetic field that simply produce small dipolar couplings, in combination with relaxation measurements, a more likely explanation is that the net orientation of the PT-linker in Cex aligned by Pfl phage is small due to its conformational mobility. Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 78 Figure 3.6. Heteronuclear 'H-f^N} NOE measurements of 15N-Thr-labelled non-glycosylated and glycosylated Cex. Heteronuclear 'H-{15N} NOE measurements of selectively 15N-Thr-labelled Cex in its (A) non-glycosylated and (B) glycosylated forms reveal that the PT-linker Nis conformationally mobile on the sub-ns timescale and that glycosylation partially dampens this mobility. Small or even negative heteronuclear 'H-{15N} NOE values indicate increased mobility on this timescale. In contrast to the threonines from the structured catalytic and cellulose-binding domains, those from the PT-linker were not specifically assigned and thus are alphabetically labelled (see Figure'3.6), without any implied correlation between corresponding residues in the two forms of Cex. Missing data points correspond to threonines that are unassigned and/or poorly resolved. Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 79 3.3.2 Glycosylated Cex expressed from S. lividans 3.3.2.1 Production of glycosylated Cex To examine the effects of glycosylation on the structure and dynamics of Cex, S. lividans was used as a surrogate expression host. Unfortunately, a uniformly 15N-labelled sample of glycosylated Cex was not prepared due to poor protein expression by this bacterium grown in M9 minimal, medium. However, using a synthetic medium supplemented with I5N-threonine (Muchmore et al., 1989), dispersed mycelial growth of S. lividans was achieved, leading to the production of glycosylated 15N-Thr-labelled Cex. Based on MALDI-TOF mass spectrometry, the expressed Cex was heterogeneously glycosylated, with an average molecular mass of 51173 Da (range, ~ 48 to ~ 53 kDa), corresponding to an average of - 25 hexose residues per protein molecule. 3.3.2.2 Independence of the catalytic and cellulose-binding domains with PT-Linker glycosylation The 'H- 1 5 N TROSY-HSQC spectrum of glycosylated 15N-Thr-labelled Cex is shown in Figure 3.5 B. Similar to that of the non-glycosylated protein, this spectrum consists of a set of well-dispersed peaks, assignable to the catalytic and cellulose-binding domains. When compared to the spectrum of the non-glycosylated full length Cex, it can be seen that the domains remain independent as evidenced by the lack of chemical shift perturbations. The glycosylated linker, therefore, does not induce any domain interactions in Cex (Figure 3.7). Only Thr3 showed a minor shift difference (~ 11 Hz) and this can be attributed to its spatial proximity to the C-terminus of the catalytic domain and hence to the. glycosylated linker. Overall, the lack of any significant chemical shift perturbations demonstrates that glycosylation of Cex does not alter the structural environments of the threonines within either domain in the full length protein. This also confirms previous studies demonstrating that glycosylation occurs exclusively within the PT-linker, and not in the structured domains of Cex (Ong et al., 1994). This comparison, however, was determined from a more limited sampling of amide residues, as the full length glycosylated Cex can only be expressed specifically-labelled with 1 5 N-Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 80 12 i 10 -x i 1 1 — . 1 CexCD CexCBD Residue Number Figure 3.7. Amide chemical shift perturbation between 15N-Thr-labelIed non-glycosylated and glycosylated Cex. The calculated amide chemical shift perturbation (in Hertz) between the specifically I5N-threonine labelled full length glycosylated Cex (from S. lividans) and the specifically 15N-threonine labelled non-glycosylated Cex (from E. coli) is shown. The lack of significant shift perturbations show that the presence of a glycosylated linker has no structural effects pertaining to the relationship between the two Cex modular domains in solution. Data corresponding to overlapped and/or unassigned peaks are not plotted. Note that the typical 'H line width of an amide signal in these spectra is ~ 35 Hz with data processed to 5 Hz/point. For comparison, formation of a trapped glycosyl-enzyme intermediate leads to amide chemical shift perturbations in excess of 100 Hz (Figure 2.3). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 81 threonine. Therefore, all of the comparisons made with the non-glycosylated full length Cex are only possible between corresponding threonines. Furthermore, based on 1 5 N T] and T 2 relaxation measurements for a total of 11 and 13 threonines, respectively, the effective xc values for the catalytic and cellulose-binding domains in glycosylated 15N-Thr-labelled Cex (~ 0.4 mM) were determined to be 21.3 ± 1.1 and 18.3 ± 0.6 ns. As expected, these values are higher than those measured for the unmodified protein due to the increased average molecular mass of ~ 4 kDa from the added sugars, possibly combined with reduced linker flexibility (discussed below). However, the fact that the two domains exhibit significantly different diffusion properties indicates that they still tumble semi-independently while tethered by the glycosylated PT-linker. 3.3.2.3 Conformationally dynamic and predominantly unstructured glycosylated PT-Linker After accounting for the threonines in the catalytic and cellulose-binding domains, the remaining peaks in the 'H- I 5 N TROSY-HSQC spectrum of 15N-Thr-labelled Cex from S. lividans must arise from the glycosylated PT-linker. These include a cluster of strong signals with random coil *H N chemical shifts. Several weak signals, including two peaks with amide shifts outside of the random coil range (labelled T-L-l and T-L-m in Figure 3.5 B) are also detected, indicative of heterogeneous glycosylation and/or cis-trans Thr-Pro isomerization. Due to this post-translational modification, the chemical shifts of the linker threonines differ in the HSQC spectra of glycosylated versus non-glycosylated Cex. Along with the lack of specific assignments, this precluded a residue-by-residue comparison of the interdomain sequence in the two forms of Cex. Nevertheless, the observation of random coil chemical shifts demonstrates that the PT-linker remains predominantly unstructured upon glycosylation. The more dispersed : H N chemical shifts of two of the threonines may reflect local conformational effects due to the attached hexose moieties. Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 82 The dynamic properties of glycosylated N-Thr labelled Cex were also investigated by N relaxation measurements (Figure 3.6 B). Similar to the non-glycosylated form of the protein, threonines within the well-structured catalytic and cellulose-binding domains exhibited uniformly high 'H-{15N} NOE values. In contrast, PT-linker threonines showed reduced ^ -{^N} NOE values, indicative of significantly greater flexibility on the sub-ns timescale. However, these values were consistently positive and higher than those observed with unmodified Cex (Figure 3.6 A). Furthermore, fitting of the relaxation data measured for 4 linker threonines with strong, resolved signals yielded an average effective r c of ~ 8 ns and an average S2 of ~ 0.8. Although complicated by the heterogeneous levels of modification, these values are clearly higher than those found for unmodified Cex, thus demonstrating that glycosylation partially dampens the fast timescale mobility of the PT-linker.' Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 83 3.4 DISCUSSION 3.4.1 Cex is composed of independent catalytic and cellulose-binding domains tethered by a flexible PT-Linker In this chapter, NMR spectroscopy was used to characterize native Cex in its non-glycosylated and glycosylated forms. In particular, this represents the first direct conformational study of the PT-linker from such a class of modular glycoside hydrolases. Although Cex is a 47 kDa protein, it yields excellent quality NMR spectra, likely due to the independence of its well-folded constituent domains. Based on amide chemical shift comparisons, combined with amide RDC and 1 5 N relaxation measurements, it can be demonstrated that the catalytic domain and cellulose-binding domain in Cex behave as "beads-on-a-string," joined by a flexible linker (Figure 1.4). This conclusion is supported by several lines of evidence. First, the lack of any significant chemical shift differences between corresponding amides in full length Cex versus isolated CexCD or CexCBD (separated or mixed) reveals that the catalytic and cellulose-binding domains do not significantly interact in a non-covalent manner with one another, or with the PT-linker, when tethered together in their native context. Second, the significantly different effective tc values (17.6 ± 1.1 ns versus 14.3 ± 1.0 ns) measured for the two domains in Cex indicate that each undergoes rotational diffusion semi-independently. Third, the interdomain mobility of Cex is also reflected by the distinct alignment tensors describing the differential orientation of its constituent catalytic and cellulose-binding domains by Pfl phage. Fourth, in contrast to these well-structured domains, the threonine residues within the PT-linker have random coil chemical shifts, low or negative heteronuclear 'H-{15N} NOE values, and small 'DHH RDC values. Thus, this segment of Cex is predominantly unstructured and conformationally mobile on the ns-ps time scale. Not unexpectedly, the structural organization of Cex is consistent with the functional properties of this well characterized glycoside hydrolase. In particular, the enzymatic activity of isolated CexCD towards cleavage of soluble substrates is similar to that of the full length Cex, with or Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 84 without linker glycosylation (Langsford et al, 1987; Tomme et al, 1995b). Thus, neither the linker nor the cellulose-binding module alters the catalytic properties of the catalytic domain of Cex. Likewise, the relative binding affinities of non-glycosylated Cex and the isolated CexCBD for microcrystalline cellulose (Avicel) are very similar, indicating that the catalytic domain and linker neither contribute to, nor interfere with, cellulose binding (Ong et al, 1993). Complementing these findings, thermal denaturation studies also revealed that the stabilities of the catalytic domain and cellulose-binding modules of Cex do not change significantly upon their separation (Nikolova et al, 1997). 3.4.2 Glycosylation partially dampens the fast timescale motions of the PT-linker, but does not perturb the catalytic or cellulose-binding domains The effects of glycosylation on Cex were investigated using selectively l5N-Thr-labelled protein produced in S. lividans. These efforts represent the first time that a prokaryotic expression host has been used to overexpress a glycosylated isotopically-labelled protein for NMR spectroscopic analyses. Based on their invariant threonine ' H N and 1 5 N chemical shifts, glycosylation of the PT-linker does not affect the structure of either the catalytic or cellulose-binding domains of Cex. This is consistent with the similar catalytic activities of unmodified and glycosylated Cex towards soluble substrates (Langsford et al, 1987). Furthermore, glycosylation does not induce any predominant structure in the PT-linker, as shown by the random coil shifts of its constituent threonines. The glycosylated PT-linker also remains flexible on the sub-ns timescale, as indicated by significantly lower 'H-{15N} NOE values than those measured for the threonines in the structured catalytic and binding domains. The fast timescale dynamics of the glycosylated PT-linker are, however, dampened relative to the unmodified linker, as reflected by slower effective tc and increased 'H-{I5N} NOE and S2 values compared to the unmodified linker. These observed dynamic changes, which should be viewed qualitatively due to heterogeneous levels of glycosylation, may result from increased hydrodynamic Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 85 drag from the covalently attached a-mannose residues and/or steric interactions restricting the conformational space accessible to the PT-linker. Indeed, light scattering and 1 3C-NMR spectroscopic studies have demonstrated that extensive O-glycosylation leads to a pronounced lengthening and stiffening of the polypeptide chain of ovine submaxillary mucin (Gerken et al, 1989; Shogren et al, 1989). Likewise, scanning tunelling microscopy and SAXS analyses of the linker in Aspergillus niger glucoamylase I and the "double linker" in a chimeric cellulase were significantly longer than expected for random coil glycopeptides, leading to the hypothesis that steric restraints introduced by the attached sugars drove its conformational ensemble towards more extended forms (Kramer et al, 1993; Receveur et al, 2002). The restricted flexibility of the PT-linker upon O-glycosylation may in turn reduce the relative mobility and increase the average separation of the catalytic and cellulose-binding domains of Cex. Along with an overall increase of ~ 4 kDa in mass, tmYmay lead to the slower effective xm values measured for the two domains relative to their counterparts in the unmodified protein. However, it remains to be established whether or not this alters the activity of Cex towards its natural substrates. To date, the best documented role of glycosylation is to enhance the viability of Cex in an extracellular environment by providing protection for the proteolytically-vulnerable PT-linker (MacLeod et al, 1992). In addition, the presence of glycans has been reported to marginally increase the relative affinity of a Cex fragment, consisting of only the PT-linker plus the CexCBD, for crystalline cellulose relative to its non-glycosylated counterpart. It is possible that the enhanced binding character is mediated through direct sugar-sugar interactions (Ong et al, 1994). 3.4.3 Implications for the structure of Cex The demonstration by NMR methods that Cex is composed of independent, well-folded catalytic and cellulose-binding domains tethered by a flexible PT-linker, refines the structural model for this glycoside hydrolase. From early SAXS measurements, the maximum dimension of Cex within its conformational ensemble that contributes detectably to scattering, D,™*, was reported to be Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 86 ~ 140 A (Miller et al, 1995). With consideration of the known structures of CexCD and CexCBD, this corresponds to a for the 20 residue PT-linker of - 50 to 70 A. This value is similar to that of ~ 65 A estimated for the highly related 23 residue PT-linker of CenA from a comparison of D , ^ values measured for the wild type and a deletion mutant of this C. fimi endoglucanase (Shen et al, 1991). The average of ~ 3 A/residue for these PT-linkers corresponds to a relatively extended conformation. Neighbouring C a-to-C a distances for a-helices and p-strands are 1.5 and 3.3 A, respectively (Argos, 1990), and a statistical analysis of helical and extended linkers yielded average displacements of 1.5 and 3 A/residue, respectively (George and Heringa, 2002). The central proline tripeptide observed with weak electron density in the linker of S. olivaceoviridis E-26 xylanase spans a C a-to-C a distance of 6.6 A (Fujimoto et al, 2002). The PT-linkers of Cex and CenA were initially described as extended and rigid because of the relatively, large D ^ values measured by SAXS (Shen et al, 1991). However, as discussed above, NMR spectroscopy reveals that the linker of Cex is predominantly unstructured and flexible on the ns-ps timescale. In this respect, the two techniques are complementary as SAXS provides a measure of the possible extension of a molecule, but only indirect insights into its motional properties, whereas NMR spectroscopy most readily yields local dynamic, rather than global structural, information. Based on chemical shift, 1 5 N relaxation, and RDC measurements, the fast dynamics of the PT-linker likely arise from local backbone mobility; however, concerted motions of segments of the linker may also occur. NMR studies have indicated that proline/alanine-rich peptides, corresponding to sequences in the light chain of skeletal myosin and in the pyruvate dehydrogenase complex, are not simple random coils, but rather preferentially adopt extended conformations with partially restricted flexibility (Bhandari et al, 1986; Radford et al, 1989). With a cyclized sidechain, prolines disfavor a-helices due to their inability to act as hydrogen bond donors, combined with steric interactions of their C 8 H 2 group that bias the dihedral angle of the preceding residue to the p-sheet region (i|/ ~ +135°) of the Ramachandran plot. At the same time, with a § dihedral angle constrained near -65°, Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 87 prolines cannot adopt an anti-parallel P-strand conformation (<|> ~ -140°) (Williamson, 1994). Thus, it was proposed that Cex adopts an ensemble of linker conformations to yield a distribution of orientations and spacings between its catalytic and cellulose-binding domains; however, more extended structures likely predominate due to backbone conformational restrictions imparted by the alternating proline residues. Glycosylation may further bias this distribution towards elongated states. It is instructive to compare the properties of the C. fimi PT-linkers with those reported for the glycosylated serine/threonine-rich linkers in H. insolens. Cel45 (36 residues) and a chimera constructed from Cel6A and Cel6B of this saprophytic fungus (88 residues) (von Ossowski et al, 2005; Receveur et al, 2002). Significantly shorter displacements of - 1.4 and ~ 0.7 A/residue were estimated from the SAXS-determined Dma X values of these linkers. Furthermore, detailed fitting of the scattering profile of Cel6A/B revealed that the chimera adopts an ensemble of structures with varying interdomain spacing and that the flexible linker adopts a nonrandom distribution of conformations, with a preference for more compact states. Such compact states may be less favored with PT-linkers due to their higher content of proline residues. 3.4.4 Implications for catalysis by Cex Consistent with previous enzymatic, ligand binding, and thermal denaturation studies, our current NMR spectroscopic analyses demonstrate that Cex is composed of structurally-independent' catalytic and cellulose-binding domains, tethered by a flexible proline/threonine linker. This model, summarized in Figure 1.4, supports the hypothesis that the cellulose-binding domain targets Cex to crystalline regions of cellulose within the plant cell wall (Jervis et al, 1997; Tomme et al, 1995a)) and thereby enhances the activity of the catalytic domain by maintaining its local concentration and prolonged association with nearby hemicellulose (xylan) chains (Black et al, 1997). Simple modelling suggests that the distance between the active site of Cex and the edge of its cellulose-binding domain can range from ~ 20 A to 80 A as the end-to-end distance of the flexible PT-linker increases. This would correspond to ~ 6 xylobiose units that could be cleaved by a caterpillar-like Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 88 motion of Cex with its cellulose-binding domain anchored at a fixed position. The possible range of motion allowed by the PT-linker, particularly when glycosylated, may be restricted stemming from its preference for extended conformations. The ability of the binding domain to diffuse along the surface of cellulose (Jervis et al, 1997) would then allow the progressive hydrolysis of more distant hemicellulose chains. The cellulose-binding domain may also contribute by disrupting weakly ordered regions of the cellulose fibers (Gilkes et al, 1993). Chapter 3 - Flexibility of the Inter-Domain Linker in Cex 89 3.5 CONCLUSION In conclusion, this study definitively demonstrated that PT-linkers o f the type found in Cex are flexible, albeit less so in their glycosylated state. These results complement recent S A X S studies on cellulases with linkers o f lower proline content, which demonstrate an ensemble o f interdomain spacing and thus imply that the linker is flexible (von Ossowski et al., 2005; Reveveur et al., 2002; Violo t et al., 2005). Together, these data support a model o f modular cellulase/hemicellulase action in which the cellulose-binding domain serves as an anchor point on the plant cell wal l , while the catalytic domain performs polymer hydrolysis via a caterpillar-like motion that is coupled with diffusion o f the binding domain on the cellulose surface. A linker sequence of the appropriate length and/or flexibility is clearly crucial to this mode o f action. Dissecting the structural and dynamic properties o f Cex is key to the understanding of its mode o f action on extracellular crystalline substrates. Macroscopically, knowing that the Cex catalytic and cellulose binding domains are tethered together with a flexible linker, I w i l l now turn my attention to study the underlying microscopic properties which govern substrate hydrolysis by the Cex catalytic domain, including the pH-dependent ionization o f active-site residues in Chapter'4, and the possibility o f a -3 glycone binding subsite in Chapter 5. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 90 Chapter 4 Electrostatic Interactions and Catalysis by Cex Chapter 4 - Electrostatic Interactions and Catalysis by Cex 91 4.1 INTRODUCTION 4.1.1 Electrostatic interactions in proteins In enzymes, a select group of amino acid sidechains and/or co-factors are available to function as nucleophiles, electrophiles, or general acid-base catalysts. Their abilities to fulfill these roles at a given pH depend highly on their ionization states, as reflected by their pKa values (as defined in Equation 4.1) according to the acid-base equilibrium shown in Scheme 4.1. V (AH 3o+XAA-) [H 3Q+][A-] K„ = « — Equation 4.1. ( a H A ) [HA] H A + H 2 0 . H 3 0 + + A- Schemed. When these ionizable amino acids are present on the surface of structured proteins, and hence exposed to bulk solvent, their pKa values are mostly modulated by the ionic strength and the dielectric of the surrounding medium and the presence of other nearby charged or hydrogen-bonding groups. As a result of this aqueous environment, their pKa values are typically only slightly perturbed (< 2 units) from that of the same residue in a random coil polypeptide (Table 4.1). In contrast, the burial of titratable groups within the interior of a protein or in an enzyme active site can significantly perturb their pKa values (> 2 units) due to charge-charge and charge-dipole interactions, as well as Born or desolvation effects within a low dielectric medium (Harris and Turner, 2002). These perturbations can allow active site residues to contribute to catalysis at biologically relevant pH conditions. The ionization of catalytic groups is affected by charge-charge interactions. These can be described using Coulomb's Law (Equation 4.2), where the electrostatic energy (AE) between the two interacting charges (qi and q2) is modulated by the distance (r) relative to infinite separation and the dielectric constant of the medium (D) between them. Logically, the smaller the distance between the point charges, the greater the electric force of these attractions or repulsions. Likewise, the placement Chapter 4 - Electrostatic Interactions and Catalysis by Cex 92 Group Observed pKa ( a ) a-Amino 6.8-8.0 a-Carboxyl 3.5-4.3 B-Carboxyl (Asp) 3.9-4.0 y-Carboxyl (Glu) 4.3-4.5 5-Guanido (Arg) 12.0 8-Amino (Lys) 10.4-11.1 Imidazole (His) 6.0-7.0 Thiol (Cys) 9.0-9.5 Phenolic hydroxyl (Tyr) 10.0-10.3 Table 4.1. Random-coil pKa values of proteins. The intrinsic random-coil pKa values of ionizable groups found in proteins as tabulated by Creighton (Creighton 1993). (a)The observed values were derived from different model compounds used to represent isolated amino acid residues. The values for the terminal a-amino and a-carboxyl groups vary depending on the types of residues tested. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 93 of these charges in a low dielectric environment (e.g. the interior of a protein) will significantly increase the strength of this interaction (Creighton, 1993). A E = qiS_2_ Equation 4.2 Dr Due to electrostatic interactions, the ionization equilibrium of a titratable group will be dependent upon other charged species in its vicinity with pKa values changing in order to minimize unfavourable like-charge repulsions and to maximize favourable opposite-charge attractions. In the case of two nearby Asp or Glu sidechains, ionization of one group will increase the pKa of the other group to avoid unfavourable Coulombic interactions. This effect has been well-documented in the Bacillus circulans xylanase (Bex) (Mcintosh et al, 1996). Prior to substrate binding, the general acid Glul72 has a pKa value of 6.7 due to charge repulsion by the nearby (5.5 A) nucleophilic Glii78 (pKa 4.6). Upon neutralization of the Glu78 in the glycosyl-enzyme intermediate, the pKa value of Glul27 drops to 4.2, allowing it to act as a general base (Figure 4.1). Other less dramatic pKa perturbations have also been reported for pairs of proximal carboxyl groups in ribonuclease HI (Oda et al, 1994), hen egg-white lysozyme (Parsons and Raftery, 1972), and penicillopepsin (Hsu et al, 1977). In a similar fashion, when both proximal groups are histidines, lysines, or arginines, the pKa of one of the groups will decrease, leading to its neutralization via the loss of a proton. This is exemplified by the neighbouring lysines found in acetoacetate decarboxylase, where a proximally-located positively-charged Lysll5 drives the decrease in the pKa of the catalytic Lysll6 to facilitate formation of a Schiff base intermediate (Frey et al, 1971; Kokesh et al, 1971). When one group is positively-charged while the other is negatively-charged, the positive group will tend to lower the pKa of the negative group, and vice versa, to ensure the most favourable electrostatic interactions. As a result, perturbed pKa values of ionizable groups can be observed, such as in alanine racemase, where the general base Tyr265 has a pKa of 7.2 (a drop of ~ 2.5 units) (Sun and Toney, 1999) and in UDP-galactose 4-epimerase, where the pKa of Tyrl49 is depressed by ~ 3.5 units to become a general base for proton abstraction (Liu et al., 1997). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 94 Figure 4.1. The family 11 xylanase Bex and pKa cycling. The double-displacement mechanism employed by the family 11 xylanase Bex demonstrating the duality of Glu 172 functioning as a general acid (pKa 6.7) and a general base (pKa 4.2) during the glycosylation and deglycosylation steps, respectively. The alternating pKa value of 6.7 to 4.2, and back to 6.7, parallels the switch of Glu78 from a negatively-charged nucleophile (pKa 4.6) to a neutral glycosyl ester, is coined "pKa cycling" (Mcintosh et al, 1996). The figure is adapted from Joshi et al, 2001. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 95 pKa6.7 Glul72 Glu78 pKa4.6 pKa6.7 Glul72 I A A A / V GIu78 pKa4.6 Glul72 cr -ROH pKa 4.2 Glul72 H R 0 _ / A H » A A A A / -Glu78 F Glul72 C T ^ 0 Chapter 4 - Electrostatic Interactions and Catalysis by Cex 96 In proteins, two main types of charge-dipole interactions are present which can affect the ionization of titratable groups. Firstly, electronegative heteroatoms (usually oxygen, nitrogen, or sulfur) can form hydrogen bonds with adjacent electropositive hydrogens. The resultant "typical" hydrogen bonds have donor-to-acceptor bond lengths from 2.7 to 3.0 A, and contribute net favourable formation energy of ~ -1 to -3 kcal/mol with a protein relative to water (Harris and Turner, 2002). Accordingly, the pKa of the hydrogen bond donor and the acceptor can be decreased of increased, respectively. In situations where the heteroatomic donors and acceptors have closely-matched pKa values, very short (< 2.5 A) and very strong (10 to 20 kcal/mol) "low-barrier hydrogen bonds" can be formed (Cleland and Kreevoy, 1994). Secondly, the placement of titratable groups near macroscopic dipoles, such as the N-termini and C-termini of a-helices, can also lead to pKa perturbations due to the "helix-dipole effect" (Hoi et al, 1978; Lodi and Knowles, 1993). This macroscopic dipole arises from the parallel alignment of peptide bonds along the long axis of a helix, such that the 5+C=08" points towards the C-terminus, while the 8"N-H 8 + points towards the N-terminus. .Using triosephosphate isomerase as an example, the pKa of His95 (located at the N-terminus of a short. helix) is decreased by greater than 2 units, whereas the pKa of His 103 (located at the C-terminus of the same helix) increased by 0.6 units when compared to their respective pKa values in the unfolded protein (Sancho et al, 1992). The Born or desolvation effect results from the energetically unfavourable process of transferring a single charged group from a high dielectric environment (i.e. water, e = 78) to the hydrophobic interior of a protein where the dielectric constant is much lower. When buried, the neutral uncharged forms of a titratable group will be preferred. For example, the pKa of buried Asp99 is estimated to be greater than 9 (vs. a random-coil pKa value of ~ 4, Table 4.1) in ketosteroid isomerase (Thornburg et al, 1998), while the pKa values of the catalytic lysines in antibody aldolases can range from 5.5 to 6.0 (vs. typical values of ~ 10.8, Table 4.1) (Barbas et al, 1997). Thus, isolated, buried charge groups are energetically unfavourable; however, buried charged pairs can contribute Chapter 4 - Electrostatic Interactions and Catalysis by Cex 97 significantly to protein folding due to strong electrostatic forces overcoming the unfavourable Born effects. Regardless, if charged groups are exposed to a high or low dielectric medium, the ionic strength of the aqueous environment can modulate these dielectric effects as described by the Debye-Hiickel equation (Equation 4.3), where the effective dielectric constant (Defl) increases over that of water (DH2O) with increasing distance (d) between the interacting charges with K being proportional to the square root of the ionic strength. Equation 4.3. When small diffusible ions such as Na+ and CI" are present in water, the apparent dielectric constant of the solution increases because the ions tend to concentrate in the vicinity of charges of the opposite sign (Creighton 1993). The availability of charged ions in solution can stabilize charged sidechains to overcome desolvation, as the localized dielectric constant more closely resembles exposed aqueous conditions. In contrast, single partially-buried ionizable groups will become neutral due to desolvation effects. 4.1.2 The family 10 xylanase Cex The active site of Cex is composed of a constellation of amino acids, with many polar and/or ionizable sidechains forming a complex network of hydrogen bonds. Logically, the pH-dependent catalytic activity of this glycoside hydrolase must be modulated by these interactions. In particular, Glu233 must be negatively-charged to act as an efficient nucleophile and Glul27 must be protonated and deprotonated to act as a general acid and a general base, respectively, during this retaining double-displacement mechanism as depicted in Figure 1.1. As discussed in Chapter 1 for apo-CexCD, the pKa value of the nucleophile and general acid appear to be 4.1 and 7.7, respectively, as derived from plots of kca,/Km as a function of pH for the substrate 2,4-dinitrophenyl P-D-cellobioside (2,4-DNPC) (Figure 1.6). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 98 Under very specific conditions, the pH dependence of kcat/Km, the second-order rate constant for free enzyme and substrate, can reflect the actual pKa values of titratable groups responsible for enzyme catalysis. In order to interpret such dependences fully, the protonation equilibria involving these ionizable groups must be rapid with respect to all other steps in the reaction, and the chemical interconversion between neutral substrate and product must occur via one specific ionization state of the active site. Furthermore, if the rate-determining step of the enzyme changes with pH, then the observed apparent pKa values are derived from multiple processes and cannot be easily interpreted (Alberty and Massey, 1954). In light of these restrictions, the interpretation of the afore mentioned apparent pKa values of Cex as simply belonging to its two catalytic glutamic acid residues is tenuous. As illustrated in Figure 4.2, both catalytic groups are parts of extensive hydrogen-bonding networks, and their pKa values must therefore be perturbed by a series of interaction partners. The complexities are magnified when through-space electrostatic interactions of 5 to 10 A can also have significant impacts on pKa values. As a result, these observed apparent pKa values reflect the summation of all the complex charge-charge, charge-dipole, and desolvation interactions present. For example, when an E233D mutation was introduced in Cex, kc a t and kc a t / K m became independent of pH over the range of 4.5 to 9.0 (Figure 4.3). This specific replacement of a glutamate with an aspartate shortened the sidechain of the nucleophile, and resulted in an increase of the pKa of the general acid/base residue by at least one unit. This is unlikely to be due to simple electrostatic effects involving the nucleophile, which would give rise to the opposite response (MacLeod et al, 1996). Under similar restrictions, the pH-dependence of kc a t can yield information about the pKa values of residues involved in the rate-limiting step for an enzymatic reaction. For Cex with aryl cellobiosides, this is the deglycosylation step. Prior experiments did not show a pH-dependence of kcat over the range of 4-9, therefore, limiting our understanding of the pKa values of key residues taking part in this reaction step (Figure 1.6). As mentioned, this lack of dependence can be due to the narrow experimental pH range examined due to concerns about enzyme stability (MacLeod et al, 1996) Chapter 4 - Electrostatic Interactions and Catalysis by Cex 99 Figure 4.2. Inter-residue and inter-molecular active site interactions in apo- and 2FCb-CexCD. A schematic representation of the CexCD active site highlighting the inter-residue interactions in (A) apo-CexCD, and (B) the inter-molecular interactions between the enzyme and a covalently-bound 2F-cellobioside (green). The nucleophile (Glu233) and the general acid/base residue (Glul27) are highlighted in red. The proposed hydrogen bonds are denoted (dashed lines) along with their donor-acceptor distances (A). The ionization states at pH 6.5 for the various amino acid sidechains are derived from the work described in this chapter. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 100 Chapter 4 - Electrostatic Interactions and Catalysis by Cex 101 Figure 4.3. pH-dependence of wild-type and E233D mutant of Cex towards 2,4-DNPC. The pH-dependence of (top) k c a t and (bottom) k c a t / K m of the (•/•) wild-type and (p/o) E233D mutant of Cex for the hydrolysis of 2,4-DNPC. The figure is adapted from MacLeod et al. (MacLeod et al, 1996). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 102 (Chapter 1). However, it does suggest that pKa values of the general acid/base Glul72 decreased by at least 4 units relative to the free enzyme. 4.1.3 NMR spectroscopy and pKa determination Given the vast number of X-ray derived crystallographic structures for protein systems, and the ever-increasing speed with which closely related structures are being solved, it is unfortunate that only data collected by neutron scattering and ultra-high resolution X-ray diffraction contain information pertaining to ionization states of residues (i.e. the precise location of protons). Therefore, to avoid the ambiguities of interpreting apparent pKa values from pH-dependent kinetic experiments, NMR spectroscopy is widely used to directly interrogate the site-specific protonation states of ionizable groups based on chemical shifts, scalar (J) couplings, and pH-depehdent changes in these parameters. The direct observation of nuclei within ionizable moieties, such as the carboxyl 1 3 C y / 8 of Asp and Glu residues, the imidazole 1 5 N 5 1 / e 2 of histidines, and the amine 1 5N^ of lysines provides the most unambiguous routes to these measurements due to their large chemical shift changes upon ionization (Betz et al.,.2004; Blomberg et al, 1977; Joshi et al, 2001; Lindman et al, 2007; Lui et al, 1997; Mcintosh et al, 1996; Oda et al, 1994; Plesniak et al, 1996; Poon et al, 2006; Yu et al, 1994). However, nuclei throughout an amino acid will show pH-dependent spectral changes, generally of decreasing magnitude with increasing through-bond and through-space separation from the ionizable moiety (Bachovchin et al, 2001; Betz et al, 2004; Gao et al, 2006; Singer and Forman-Kay, 1997). Unfortunately, many challenges exist when one is trying to extract meaningful pKa information from specific residues. For example, in its protonated state, the ionizable proton is generally not observed due to the rapid hydrogen exchange (HX) with water (protons with exchange rates > -100 s"1 are generally NMR invisible (Iwahara et al, 2007)). And of course in the deprotonated state, the proton is absent from the sidechain. Therefore, an alternative involves the measurement of the pH-dependent chemical shifts of the amine or imidazole 1 5 N by insensitive direct Chapter 4 - Electrostatic Interactions and Catalysis by Cex 103 detection or multiple-bond heteronuclear correlation experiments (Blomberg et al, 1976; Knoblauch et al, 1988; Leipert et al, 1975; Pelton et al, 1993; Singer and Forman-Kay, 1997). The measurement of carboxyl 1 3 C can be achieved by employing 'H- 1 3 C heteronuclear correlation experiments to neighbouring 'H nuclei (Gao et al, 2006). For large proteins, the observation of buried amino acid sidechains can also be a challenge due to the signal broadening from dipolar couplings, which can sometimes be overcome by expressing deuterated proteins. Also, multiple nuclei may not be clearly assignable if their chemical shifts are coincidentally overlapped. Finally, on a fundamental level, the availability of chemical shift information about a nucleus of interest is not enough to deduce its ionization state and/or to extract its pKa value as chemical shifts can be significantly affected by protein structure and electrostatics. One must therefore observe significant changes in chemical shifts during pH titrations prior to drawing these conclusions. Unfortunately, it is common for proteins to become unstable or chemically reactive under extreme pH conditions. As a result, it is not always possible for titrations to span sufficiently large pH ranges in order to observe the effects of changing ionizations on chemical shifts. Therefore, the use of NMR spectroscopy is best suited for ionizable residues with pKa values near neutrality. In this Chapter, by using NMR spectroscopy, the active site titratable groups in CexCD are completely characterized in order to dissect the complex hydrogen-bonding networks present, and how these inter-relationships modulate substrate binding and catalysis. Furthermore, as an added advantage when compared to kinetic studies, pH titrations can also be performed on the covalently-inhibited CexCD to gain valuable insights into the charge states of residues poised to undergo the rate-determining deglycosylation step of the enzyme mechanism. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 104 4.2 MATERIALS AND METHODS 4.2.1 Expression and purification of uniformly and selectively isotopic-labelled CexCD The expression and purification of uniformly (U) and selectively isotopic-labelled CexCD (U-15N; U- I H/ 1 3 C/ 1 S N; 1 3C-Glu; 1 3C 8-Glu; 1 3C e l-His) were performed according to the afore mentioned protocols (Poon et al, 2007a; Chapter 2). All uniformly-labelled proteins were produced from M9 minimal media enriched with 1 g/L of (15N, 98%) NH4C1 (Stable Spectral Isotope) and 1 g/L of 1 5 N- • Celtone (Stable Spectral Isotopes), while all selectively-labelled proteins are produced from a synthetic medium (Muchmore et al, 1989) enriched with 350 mg/L of ( l 3C, 98%) L-glutamic. acid (Cambridge Isotope Laboratories Inc. (CIL)), 325 mg/L of ( 1 3C 8, 99%) L-glutamic acid (CIL), 400 mg/L of (1 5N, 98%) L-glutamic acid (CIL), or 75 mg/L of ( , 3 C e \ 99%) L-histidine-HCl-H20 (CIL). The media used to produce the two selectively 13C-Glu-labelled CexCD samples was deficient in L-glutamine and supplemented with 1 g/L (15N, 98%) NH4C1. The expression host therefore produced 13C/15NMabelled glutamine from the labelled glutamic acid (Waugh, 1996). Covalent modification of CexCD was achieved by the incubation of the enzymes with ~ 3:1 molar equivalent of 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-cellobioside (2F-DNPC) (McCarter et al, 1993). Non-covalent complexes of CexCD were formed in the presence of ~ 2:1 molar equivalent of xylobio-isofagomine (l,5-imino-l,4,5-trideoxy-3-0-(y5-D-xylopyranosyl)-D-^reo-pentitol) (Xblso) or xylobio-imidazole ((6S,7S,8S)-7,8-dihydroxy-6-(y5-D-xylopyranosyloxy)-5,6,7,8-tetrahydroimidazole[l,2-a]pyridine) (Xbhn) (Williams et al, 2000). 4.2.2 NMR spectroscopy Unless otherwise mentioned, all NMR experiments were performed on a Varian INOVA 600 MHz spectrometer equipped with a gradient triple resonance room temperature or cryogenic probe at 30 °C. Spectra were referenced to external sodium 2,2-dimethyl-2-silapentane-5-sulphonate and 1 5 N Chapter 4 - Electrostatic Interactions and Catalysis by Cex 105 shifts indirectly via gyromagnetic ratios (Wishart et al, 1995b). Reported chemical shifts were not corrected for an offset of ('jNH)/2 due to the TROSY selection, or for 2 H isotope effects. NMR data were processed using NMRpipe (Delaglio et al, 1995) and analyzed using SPARKY 3.0 (Goddard and Kneeler, 1999). The detailed assignment strategies for the lysine, histidine, and glutamic and aspartic acids residues are discussed in detail in sections 4.3.1.1, 4.3.2.1, and 4.3.3.1, respectively, while the pulse-sequences used are summarized in Table 4.2 and Figure 4.4. 4.2.3 Ionization states and pH titrations of CexCD by NMR spectroscopy The pH dependences of the titratable groups being investigated were extracted from the changes in observed chemical shifts as a function of pH. These observed changes can be a result of scalar coupling or through-space effects. A list of the methodologies used is summarized in Table 4.3. The pH of the protein samples was first increased, and then decreased in 0.2 to 0.3 unit increments-from a starting value of 6.5 by the addition of microlitre aliquots of 0.1 to 0.5 M NaOH and 0.1 to 0.5 M HC1 respectively. The lower limit for titration was pH ~ 3 to avoid protein aggregation. The apparent pKa values were determined from, fitting the measured chemical shifts as a function of pH to standard equations describing one (Equation 4.4), two (Equation 4.5), or three (Equation 4.6) macroscopic protonation equilibria (Joshi et al, 2001). 8 a10- p H+6 b10- p K a 5 0 b s = -5 b — Equation 4.4. io~ p H +io- p K a _ 5a10"2 p H +5 b10" ( p H + p K a | ) +8 c10" ( p K a ' + p K a 2 ) 5 obs - 1 Q - 2 p H + 1 0 - ( p H + p K a i ) + 1 0 - ( p K a , + P K a 2 ) Equation 4.5. 5 10~3pH +5 b10 _ ( p H + p K a , ) +5 i o _ ( p K a 2 + p K a 3 + p H ) + 5 d i o - ( p K a ' + p K a 2 + p K a 3 ) ^ o b s ~ 1()~ 3 p H + 1 Q - (pKa 3 +2pH) + jQ-(pKa2+pKa3+pH) + jQ-(pKa,+pKa2+pKa3) Equation 4.6. Errors are estimated to be ± 0.1 units for all pH measurements, while errors associated with fitting of the pKa values were obtained using the program SigmaPlot. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 106 Isotopic Labeling NMR Experiments Nuclei Assigned References U- J i N 1 5 N HMQC ± tl decoupling Lys47 Griffey et al. 1984 Grzesiek et al. 1993 U- 1 5 N 15N-editedNOESY Lys302 Cavanagh et al. 2006 DCN C(C')TOCSY-NH Lys sidechain 1 3 C Cavanagh et al. 2006 DCN C p H 8 His ' H 8 2 Yamazaki et al. 1993 CT 1 3C-HSQC Slupskyef a/. 1998 DCN H i s , 3 C 8 2 Santoroef a/. 1992 U- 1 5 N Multiple-bond 1 5N-HSQC His ' H 8 1 , 1 5 N 5 1 , 1 5 N e 2 Pelton era/. 1993 Singer et al. 1997 DCN CT 1 3C-HSQC His 1 3 C e l Santoroera/. 1992 1 3C s l-His 1 3C-CPMG-HSQC His 1 3 C 6 1 Mulder et al. 1996 DCN C 8 / Y(C pC aC')NH Glx 1 3 C 8 , Asx 1 3 C Y Tollinger et al. 2002 DCN H 2 NC 8 Gin xllz2, , 3 C 8 Farmer et al. 1996 DCN H 2NCY A s n ' H 8 2 , 1 ^ Farmer et al. 1996 DCN H 2 NC Y C P Gin ' H e 2 , 1 5 N E 2 Wittekind et al. 1993 DCN H 2 N C p C a Asn'H 8 2 , , 5 N 8 2 Wittekind et al. 1993 Table 4.2. Labelling strategies and NMR experiments used to assign resonances of ionizable groups in the Cex active site. A summary of the NMR experiments employed, with the required isotopic labeling, to assign the resonances of the various nuclei needed towards the pH titration of ionizable groups in the Cex catalytic domain. The experiments were measured for both apo- and 2FCb-CexCD at pH 6.5 and 30 °C, with the exception of the C 8 / Y(C pC aC')NH experiments (40 °C). (a)Cartoon representations of these experiments are shown in Figure 4.4 for clarity. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 107 Figure 4.4. NMR experiments used to assign resonances of ionizable groups in the Cex active site. Cartoon representations of the 1 H/ 1 3 C/ 1 5 N NMR experiments employed to assign the resonances of the various nuclei needed towards the pH titration of ionizable groups in the Cex catalytic domain. The experiments were measured for both apo- and 2FCb-CexCD at pH 6.5 and 30 °C, with the exception of the C 8 / y(C pC aC')NH experiments (40 °C). For each experiment, the detected atomic resonances are coloured in red, while the nuclei which transfer magnetization are coloured in green. For the 15N-edited NOESY experiment, the green dashed line denotes through-space NOE interactions. The complete descriptions of these experiments along with pertinent citations are located in Table 4.2. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 108 H H\l/H N 15 N H M Q C O C(C')TOCSY-NH P . a H IN X I 1 H O v / HE M "" H N N \ H H V e1 \ j? l_M^ C P H 5 51 / / V 8 2 >C 15N-edited NOESY 82 P H el H e2 51 // V 8 2 ^ H 8 2 P V Multiple-bond , 5 N - H S Q C 81 He1 H e 2 // \ C 52 C T " C - H S Q C H el H c2 51 / / \ 82 'I -H 52 X C T " C - H S Q C " C - C P M G - H S Q C r H .N. H O c 5 / Y ( c P c a c . ) N H i X o ||Y/5 N5/e H H 2 N C O V "aVP c Y ^ C \ N ^ H 5/e H H , N C a C p Chapter 4 - Electrostatic Interactions and Catalysis by Cex 109 Isotopic Labeling NMR Experiments Reporter Nuclei Titrating Moieties U- 1 SN 'H-^N TROSY HSQC Amides Indoles Neighbours U- 1 SN 'H- 1 5N HSQC Lys 15N^ Amines I 3CE l-His 'H-^C HSQC His 'H E , 1 3 C E Imidazole 1 3C 8-Glu 1-D 1 3 C Glu 1 3 C 5 Carboxyl U- 1 3C-Glu "C^COIT 1 3 C 5 . Carboxyl Table 4.3. Labelling and pH titration strategies used to determine pKa values of ionizable groups in apo- and 2FCb-CexCD. A summary of the pH titration strategies used, with the respective isotopic labelling required, to elucidate the pKa values of the ionizable residues of interest in the Cex catalytic domain. Titrations were performed on apo- and 2FCb-CexCD to gain insight into the charge states of amino acid residues in both states of the enzyme. Titrations were not possible using the U- 1 3C-Glu labelled CexCD due to the lack of resonance assignments as mentioned in Section 4.3.3.2.3. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 110 4.3 RESULTS 4.3.1 Lysines 4.3.1.1 Lysine NMR assignments In the 'H- 1 5N-HMQC spectrum (Griffey et al, 1984; Grzesiek et al, 1993) of uniformly-labelled 15N-CexCD, recorded with an atypical upfield 1 5 N spectral window, a single peak at chemical shifts characteristic of an amine ('Hc 8.1 ppm, 1 5N^ 34.8 ppm) was observed (Figure 4.5 A). On the basis of a 3-D 15N-edited NOESY experiment (Cavanagh et al, 2006) as well as the previously obtained NMR assignments for CexCD (Poon et al, 2007a; Chapter 2), this signal was attributed to the amine of Lys302. Subsequently, covalently modifying CexCD using 2F-DNPC gave rise to a second amine signal observed at 8.0 ppm (1H(;) and 35.6 ppm (15N^) (Figure 4.5B). This signal was also observed upon the addition of the noncovalent inhibitors xylobio-isofagomine (Xblso-CexCD) and xylobio-imidazole (Xblm-CexCD), albeit with slightly differing chemical shifts (Figure 4.6). Based on available structural information, this signal was assigned to the lone lysine, Lys47, in the active site of CexCD (Notenboom et al, 2000; White et al, 1996). In an attempt to further confirm the assignment for Lys47, a uniformly-labelled deletion mutant of CexCD (K47A) was expressed. Unfortunately, the activity of this mutant towards 2F-DNPC was found to be so low that it could not be trapped in its glycosyl-enzyme intermediate form. However, a signal from Lys302 .was still detected. These assignments (Appendix 1 and Appendix 2) have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under the accession numbers 7264 and 7265 for apo-and 2FCb-CexCD, respectively. 4.3.1.2 Lysine ionizations and pH-dependent titrations The ionization state of the observable CexCD and 2FCb-CexCD lysines (Lys302, Lys47 and Lys302, respectively) at pH 6.5 and 30 °C can be unambiguously determined by the analysis of their one-bond ! H- 1 5 N coupling patterns found in 'H- 1 5N HSQC experiments without 'H-decoupling during Chapter 4 - Electrostatic Interactions and Catalysis by Cex 111 apo-CexCD 2FCb-CexCD pH 6.5 30 °C B j-25 K302 , , 'PNk M 8 , 6 8 4 8 2 8 0 7 8 7 6 1-35 pH 6.5 t 30 °C i l l pH 6.5 10°C D ;,K47 pH 6.5 10 °C 25 30 E Q. CL 8.0 7.5 pH 5.6 10 °C 7.0 1 H (ppm) ,.,K47 8.0 7.5 pH 5.6 10 °C 35 ! 25 30 35 7.0 Figure 4.5. ' H - ^ N H M Q C spectra of apo- and 2FCb-CexCD at varying pH and temperature conditions. 'H- 1 5 N HMQC spectra of (A, C, E) apo-CexCD and (B, D, F) 2FCb-CexCD. Samples contained ~ 0.4 mM uniformly 15N-labelled protein in 20 mM potassium phosphate and 0.02% NaN3. The gradient HMQC sequence incorporated selective flipback pulses to minimize water excitation (Griffey et al.,. 1984; Grzesiek and Bax, 1993), and delays set for I'JNHJ ~ 75 Hz. Sensitivity enhanced flipback HSQC experiments optimized for AX 3 spin systems yielded comparable spectra, albeit with marginally less signal-to-noise, for this 34 kDa protein. The high-resolution 'H-coupled 'H- 1 5N HSQC spectrum of 2FCb-CexCD at 30 °C and pH 6.5 is shown, along with the l5tf traces at the 'H c shifts of K47 and K302 (B, inset). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 112 in 30.0 32.0 34.0 36.0 38.0 40.0 30.0 32.0 £ 34.0 Q. QL 36.0 38.0 40.0 30.0 32.0 34.0 36.0 38.0 40.0 A Lys302 OH HO—r-~J~-r~~0. o - ^ - r - V H HO ' I E233 K47 B Lys302 UN H O — y - ^ ^ - ^ r ^ Q - ^ V - - —"V K47 OH o t £ ^ p c Lys302 K47 8.4 8.2 8.0 7.8 7.6 7.4 7.2 1H (PPm) Figure 4.6. 'H- 1 5 N HMQC spectra of 2FCb-CexCD, Xblso-CexCD, and Xblm-CexCD. The 'H-1 5 N HMQC spectra at pH 6.5 and 30°C of (A) 2FCb-CexCD, (B) Xblso-CexCD, and (C) Xblm-CexCD showing the lysine *H^ and 1 5N 1' region. The buried structural Lys302 is unperturbed with the addition of the different inhibitors, while the active site Lys47 is affected. The xylobio-isofagomine (Xblm) inhibitor is assumed to be positively-charged (Schubert et al, 2007). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 113 the tl period. The 1 5N^ signals of Lys47 and Lys302 in 2FCb-CexCD, as well as Lys302 in apo-CexCD, appeared as quartets split by | % H | ~ 75 Hz (Figure 4.5 B-inset). Thus, each nitrogen is directly bonded to three protons and both lysines are in their ionized (-NH3+) states at pH 6.5. Furthermore, the chemical shifts of the two residues did not change significantly between pH 3 (below which CexCD aggregates) and pH 9 (above which rapid HX preludes their detection) (Figure 4.7 A, B). Given the lack of an observable titration, the pKa values of Lys47 and Lys302 must be > 9 in the glycosyl-enzyme intermediate, while the same behaviour was observed for Lys302 in unmodified CexCD (data not shown). As a reference, 1 5 N NMR pH titrations yielded a pKa of 11.1 for the side chain amine of ^N'-lysine (Isotec), with 1 5N^ chemical shifts of 32.5 and 25.5 ppm in its ionized (-NH3+) and neutral (-NH2) states, respectively (at 25°C) (Figure 4.7 C). A summary of the obtained pKa values, for the Lys47 and Lys302 in CexCD and the '^-labelled lysine standard, is presented in Table 4.4. CexCD contains 18 lysines, most of which are exposed on the surface of the protein and thus not detected in a 'H- 1 5 N HMQC spectrum recorded at 30°C and pH 6.5. This lack of observation results from rapid HX, which leads to increased proton line widths and decreased efficiency of 'H- 1 5N coherence transfer. Strikingly, upon slowing exchange by simply reducing the sample temperature to 10 °C, ' H ' - 1 5 ! ^ signals from at least eight additional amines became detectable (Figure 4.5 C, D). In contrast, the peaks from the internal Lys47 and Lys302 diminished in intensity due to the slower global tumbling of the enzyme. Furthermore, since this exchange is both specific and general base catalyzed (Blomberg et ai, 1976; Englander and Kallenbach, 1983; Henry and Sykes, 1995; Knoblauch et al, 1988; Leipert and Noggle, 1975; Leipinsh and Otting, 1996), reduction of the sample pH to 5.6 led to the detection of at least 10 amines with increased signal intensities (Figure 4.5 E, F). Each of these yielded a 1 5 N quartet in a 'H-coupled HSQC spectrum and must arise from a fully protonated mobile surface lysine or the N-terminal amine. The observed chemical shift dispersion of 35.6 to 28.5 ppm in 1 5 N and 8.1 to 6.4 ppm in 'H reflects the local environments of these Chapter 4 - Electrostatic Interactions and Catalysis by Cex 114 Figure 4.7. 'H^ - and 15N -^observed pH titrations of Lys47 and Lys302 in apo- and 2FCb-CexCD. The (A) ' H 5 and (B) ' ¥ chemical shifts of Lys47 (•) and Lys302 (•) in 2FCb-CexCD are pH-independent over the pH range examined (30 °C). Unfortunately, the resonances for both lysines cannot be observed above pH 9 due to base-catalyzed exchange broadening. Since both lysines are positively-charged at pH 6.5, their pKa values are > 9. The pH titration curve for (C) '^-labelled lysine, with its pKa at 11.1 (25 °C), is shown as a reference (Poon et al., 2006). Chapter 4 - Electrostatic Interactions and Catalysis by Cex a 15 8.3 8.2 I. 8.1 8.0 7.9 7.8 A • 1H ;-Lys47 • 1H ;-Lys302 34.0 32.0 £ 30.0 a. a . Z 28.0 in 26.0 24.0 40.0- B 38.0 E Q. 36.0 • * ••••••*• *•• • «• z in 34.0-• l 5N e-Lys47 32.0- " 1 V -Lys302 pKa = 11.1 15N;-Lysine 4.0 6.0 8.0 10.0 12.0 PH Chapter 4 - Electrostatic Interactions and Catalysis by Cex 116 Residue Form Charge/Tautomer pKa pH 6.5 Lys47 apo positive >9 2FCb positive >9 Lys302 apo positive >9 2FCb positive >9 Table 4.4. Summary of the charge state and pKa values limits of the two observable Lys residues in apo- and 2FCb-CexCD. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 117 amines, rather than differences in their charge states. Unfortunately, no resolved signal was present in the spectra of apo-CexCD, yet absent in those of 2FCb-CexCD or the K47A mutant, which could be confidently attributed to Lys47 in the unmodified protein. 4.3.2 Histidines 4.3.2.1 Histidine NMR assignments Based on the reported main chain nuclei assignments for the five histidine residues (Poon et al, 2006; Chapter 2), resonances from the imidazole rings in apo- and 2FCb-CexCD were assigned using a set of heteronuclear correlation experiments. From their known 1 3 C P resonances, assignments of their imidazole ring ' H 5 2 were obtained via a C P H 8 spectrum (Slupsky et al, 1998; Yamazaki et al, 1993), and subsequently, to their 1 3 C 8 2 from a constant-time 1 3C-HSQC spectrum (Santoro and King, 1992). Signals from non- labile ' H 5 2 and 'FT1 were correlated to one another and to the neighbouring 1 5 N 8 1 and ^N 6 2 by multiple-bond 1 5N-HSQC spectra (Pelton et al, 1993; Singer and Forman-Kay, 1997) of 15N-labelled proteins (Figure 4.8). The 'FT31 was then connected to the directly-bonded 1 3 C E l in a constant-time 1 3C-HSQC spectrum in the uniformly 13C-labelled protein, as well as in a 1 3 C -CPMG-HSQC spectrum of CexCD selectively labelled with 13C£l-histidine (Figure 4.9). These NMR assignments were confirmed using spectra recorded with the apo- and inhibited forms of CexCD containing alanine substitutions at His80 or His205. These assignments (Appendix 1 and Appendix 2) have been deposited in the BioMagResBank (http://www.brnrb.wisc.edu/) under the accession numbers 7264 and 7265 for apo- and 2FCb-CexCD, respectively. 4.3.2.2 Histidine ionization states and pKa measurements The multiple-bond experiment (Figure 4.8), as mentioned above, can define the charge states and tautomeric forms of imidazole rings found in proteins (Pelton et al, 1993). The observed patterns in apo- and 2FCb-CexCD at pH 6.5, based on 2Jm and 3JNH couplings, showed that His85 is neutral and exists in the common N e 2 tautomeric state. In contrast, Hisl07, Hisl 14, and His205 are Chapter 4 - Electrostatic Interactions and Catalysis by Cex 118 H N ^ N H p o s i t j v e ( + ) HN N Neutral (N^H) N ^ N H Neutral (NS1H) apo-CexCD 2FCb-CexCD H80 +* N 5 ,H H85 N^H N^H H107 + + H114 + + H205 + + E Q . Q . 8.0 7.5 7.0 1H(Ppm) Figure 4.8. Assignment of the protonation and tautomerization states of the histidines in apo-and 2FCb-CexCD. The assignment of the protonation and tautomerization states of the five histidine residues in 15N-labelled (A) CexCD and (B) covalently-inhibited 2FCb-CexCD at pH 6.5 and 30 °C from multiple-bond 1 5N-HSQC spectra (Pelton et al, 1993). Signals near 170 ppm arise from 1 5 NH moieties, whereas those near 250 ppm are from. 1 5 N groups. The upfield-shifted peaks involving the "H 8 2 of Hisl 14 at 3.79 ppm are not shown. In addition, a summary of the predominant charge-states • of the five histidines in apo- and 2FCb-CexCD are tabulated (Schubert et al, 2007). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 119 A A H114 H H107 H20S Has H80 0 H85 9 H20S R 0 H MOT S hH H O X ^ H205 F I 0 » 1 , © H8S HBO OH x H B H20S 0 H8S H80 o o 8.5 8.0 7.5 7.0 6.5 8.5 8.0 7.5 7.0 6.5 1 H (ppm) Figure 4.9. 1 3 C-CPMG-HSQC spectra of ,3Ce,-His-labeIIed apo-, 2FCb, Xblso-, and Xblm-CexCD. The assigned one-bond 1 3C-CPMG-HSQC spectra of selectively 1 3CE l-His (A) apo-CexCD, (B) covalently-inhibited 2FCb-CexCD, and CexCD non-covalently inhibited with (C) xylobio-isofagomine (assumed to be positively-charged) and (D) xylobio-imidazole, at pH 6.5 and 30 °C (Schubert etal, 2007). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 120 positively charged. For His80, although the characteristic pattern associated with a positively-charged histidine is observed, its signal intensities are significantly lower than those of the other charged residues. Along with its chemical shifts, this indicates that His80 is partially in a neutral state. In contrast, His80 is in its neutral H 5 1 tautomeric state when Cex is covalently-inhibited. The pKa values (or limits thereto) of the five histidines in apo- and 2FCb-CexCD were determined from the pH-dependence of their I 3 C e l and ' H e l shifts (Figure 4.10). These shifts were measured using selectively 1 3 C e l His-labelled protein, for which well-resolved 1 3C-HSQC spectra can be obtained within minutes (Figure 4.9). This approach (Lui et al, 1997; Yu and Fesik, 1994) is significantly more sensitive than direct l 5 N detection (Blomberg et al, 1977; Plesniak et al, 1996) or indirect 2- or 3-bond 1 f r S 2 / e l - 1 5 N 8 , / s : 2 correlation experiments (Singer and Forman-Kay, 1997). Over the pH range of 4.3 to 10.5 for apo-CexCD, only Hisll4 (pKa 8.1) and His80 (pKa 7.9) showed measurable titrations. Similarly for 2FCb-CexCD, over a range of pH 2.8 to 10.1, only His 114 (pKa 8.1) titrated. Thus, the remaining histidines, with pH-independent chemical shifts, have pKa values that are at least 1 unit outside these ranges (i.e. given that clear spectral changes should be seen at pH = pKa ± 1). Although it is impossible to unambiguously deduce the charged states of the non-titrating histidines from their 1 3 C e l and ' H e l chemical shifts alone, this information is provided by their 1 5 N shifts (Figure 4.8). A summary of the determined pKa values and charged states for both apo- and 2FCb-CexCD is given in Table 4.5. 4.3.3 Glutamic and aspartic acids 4.3.3.1 Carboxyl NMR assignments Measurement of the pH-dependent chemical shifts of the carboxyl carbons of Glu (13C8) and Asp (13Cy) residues can define their ionization states and pKa values (Betz et al, 2004; Mcintosh et al 1996). To assign the 1 3 C 5 / y carboxyl chemical shifts for apo- and 2FCb-CexCD, a TROSY-based C 5 /*(CpC aC')NH e x p e r i m e n t (L. E. Kay, personal comm.) was run on samples of DCN-(70%-2H; Chapter 4 - Electrostatic Interactions and Catalysis by Cex 121 apo-CexCD 2FCb-CexCD E Q. CL 8.5 J 8.0 J 7.5 J 7.0 ^ . ^ S — & - 0 - S OO O O O O O I ) O O O O O O O O OOO H8S H107 •••»»•••»• H205 v H114 '••••«. H80 S ^ ^ ^ S j f c * ^ ^ ^ , * ^ . , , H W 7 H205 H114 IB E Q. Q. O CO 139 i 138 J «6J f354 i • H205 T ypi i t t i T > t i T t w HJ07 H80 • ••«« — >• • 5 a " «205 10 10 PH PH Figure 4.10. ' H E l - and , 3CE l-monitored pH titrations of 1 3 C 8 l -His labelled apo- and 2FCb-CexCD. The pH titration curves for 1 3CE l-His labelled (A, B) apo-CexCD and (C, D) 2FCb-CexCD showing the pH-dependent chemical shift changes of the (A, C) 'H 8 1 and (B, D) 1 3 C E l of each His residue (see Figure 4.9). The curves were fit to equations describing one, and when necessary, two macroscopic protonation equilibria to obtain His pKa values (Schubert et al, 2007). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 122 Residue Form Charge/Tautomer pH 6.5 pKa HisSO apo positive 7.9 2FCb neutral N 5 1 <2.8 His85 apo ' neutral N e 2 <4.0 2FCb neutral N E 2 <2.8 Hisl07 apo positive > 10.4 2FCb positive > 10.1 HI 14 apo positive 8.1 2FCb positive 8.1 His205 apo positive > 10.4 2FCb positive > 10.1 Table 4.5. Summary of charge/tautomer states and pKa values of the His residues in apo- and 2FCb-CexCD. The table is reproduced from Schubert et al, 2007. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 123 99%-13C/15N) labelled proteins at 40°C to connect the sidechain carboxyl resonances of Glx (Glu and Gln)/Asx (Asp and Asn) (i) with the corresponding amide signals of the following (/+/) amino acid residues (Figure 4.11). Due to the large size of Cex, combined with the inherent insensitivity of this experiment (which starts with 1 3 C polarization and uses several COSY-type transfers along sidechain atoms), each 3-D spectrum required ~ 4-6 days of instrument time. Out of the 40 13C-carboxyl nuclei in CexCD, signals from 30 were assigned in apo-CexCD, whereas 32 were assigned in 2FCb-CexCD. The l3C-carboxamide resonances (28 total) of Gin and Asn residues were also observed, and in similar fashion, 23 and 21 were assigned in apo- and 2FCb-CexCD, respectively. Furthermore, making use of modified H 2 NC p / Y C a / p and H 2 NC y / 8 experiments (Farmer et al'., 1996; Wittekind et al., 1993), the sidechain 1 5 N 8 / e H 2 , and 1 3 C T ' 8 assignments were obtained for Asn and Gin residues. Together, these assignments are summarized in Appendix 1 and Appendix 2 and will be deposited in the BioMagResBank (http://www.bmrb.wisc.edu/). Incomplete sidechain assignments were due to incomplete backbone 'H N - 1 5 N assignments, spectral overlap, and the presence of prolines in the i+1 positions. It is also evident that the strongest assignable signals arise from Glx and Asx resides on the surface of the enzyme, where local mobility give rise to narrower line widths for the carboxyl carbons (Chandrasekhar et al., 1994). For buried sidechains, correlation times are dominated by the global tumbling of the protein, leading to rapid T 2 relaxation, and hence signal loss, especially for large systems like CexCD. In an effort to combat these effects, data collection was carried out at an elevated temperature (40 °C), which showed significant improvements compared to the initial spectra recorded at 30 °C (data not shown). However, the free enzyme showed moderate aggregation after the 5 day duration of the experiment. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 124 apo-CexCD 2FCb-CexCD Asp123 1 3 C r Glu127 "C* Glu233 1 3 C 5 Asp277 1 3 C T Q. O- iso o Glu43 Asp123 13CY Glu127 1 3 C S Glu233* 1 3 C J Asp235 !3 C r Asp277 [ i 3 C r Figure 4.11. 1 3 C S / Y assignments of carboxyls in apo- and 2FCb-CexCD using C%(C pC aC')NH experiments. Slices from the C 8 / Y(C pC aC')NH experiments, displaying the 1 3 C 5 / Y , - H i+i crosspeaks for selected carboxyl i carbons in (left) apo-CexCD, and (right) 2FCb-CexCD at pH 6.5 and 40 °C. The slices are taken at the 1 5 N frequencies of the i+1 residues. Glu233 in 2FCb-CexCD is covalently attached to the inhibitor. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 125 4.3.3.2 Glutamic and aspartic acid ionization states and pKa measurements 4.3.3.2.1 Direct Glu 1 3 C 8 titration In similar fashion tb the Family 11 xylanase Bex (Mcintosh et al, 1996), a sample of specifically 13C5-labelled CexCD was produced for its subsequent pH titration with monitoring by one-dimensional 1 3C-NMR. 1 3C-NMR spectra were obtained as a function of pH for both selectively 1 3C 8-Glu apo- and 2FCb-CexCD. From the spectra shown at various pH conditions (Figure 4.12), it is evident that the signal-to-noise levels for the observed 1 3 C 8 peaks are weak, and the individual 1 3 C -carboxylate signals are not sufficiently resolved to allow for their successful identification based on the available 1 3 C 8 assignments. This is especially a challenge when these resonances shift with changing pH during the course of a titration. Thus for apo-CexCD and 2FCb-CexCD, none of the Glu residues could be monitored over a pH range sufficient for their pKa determination. 4.3.3.2.2 Through bond detected 1 3 C 5 titrations In light of the difficulties highlighted in the direct detection of carboxyl groups, an alternative approach was to make use of two-dimensional ^C^'FT and uO-xYp correlation experiments. This provides carboxyl 1 3 C 5 / Y shift information, indirectly detected through the adjacent sidechain aliphatic XYP® of Glu/Asp, and has been used successfully in the cases of several proteins (Betz et al, 2004; Jeng and Dyson, 1996; Lindman et al, 2007; Oda et al, 1994). Thus, a 1 3C 5(CY)H y experiment was performed on a selectively 1 3 C / 1 5 N Glx-labelled apo-CexCD sample at 30 °C. The resultant spectrum yielded only 7 (likely surface exposed residues) out of a possible 28 crosspeak partners (14 Glu and 14 Gin residues), and these could not be assigned due to their poor spectral dispersion. Uniform deuteration could not be used to minimize dipolar coupling for the buried residues as these two-dimensional experiments require the detection of the protiated FT nuclei. The covalently-inhibited CexCD was not examined using this methodology. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 126 1 3 C Chemical Shift (ppm) Figure 4.12. 1-D 13C5-monitored pH titration of apo-CexCD. A stacked plot of the 1-D , 3C-NMR monitored pH titration of 1 3C 5-Glu CexCD at 30 °C. Heavily-overlapping peaks and low signal-to-noise prevented unambiguous assignments and therefore reliable extraction of carboxyl pKa values. The sharp upfield peak was separately determined to be an EDTA contaminant. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 127 4.3.3.2.3 Ionization information from 1 3 C S and 1 3 C Y chemical shifts The observed 1 3 C Y and 1 3 C 5 chemical shifts can provide insights into the ionization states of specific carboxyl groups in apo- and 2FCb-CexCD at pH 6.5, but not pKa information for these groups. Unfortunately,.due to the low signal-to-noise ratio of these experiments (each requiring 4 to 6 days to record), measurements at varying pH conditions were not possible. Attempts to use a faster two-dimensional 1 3 c y / 8 / 1 H N version of the C 8 / y(C pC aC')NH experiment in pH titrations were hampered by spectral overlap. The chemical shifts of nuclei arise from the surrounding electronic environments, and while residing in random-coil or surface-exposed regions, these shift measurements can be a reliable monitor of intramolecular ionization states due to the lack of complex electrostatic influences from neighbouring charged groups and aromatic ring currents. Conversely, when the nuclei of interest form parts of enzyme active sites, it is much more difficult to interpret chemical shifts as they can be influenced by both intramolecular and intermolecular effects. These considerations are especially important when considering the complicated inter-atomic electrostatic and/or hydrophobic interactions between neighbouring amino acid residues and between the enzyme and the bound inhibitor in the Cex active site. Exercising caution, the inspection of the distribution of assigned 1 3 C y / s chemical shifts for the ionizable Asp and Glu residues revealed that most have similar downfield chemical shifts, suggesting that they are most likely negatively-charged at pH 6.5 (Figure 4.13). For the Asp residues, Aspl4 appears to be in the midst of a titrationj while Asp 123 and Asp277 exhibited much more upfield 1 3 C y chemical shifts suggestive of being protonated and neutral in both apo- and 2FCb-CexCD. This is in contrast to previously published structures (White et al, 1994; White et al, 1996), where Asp 123 was assumed to be deprotonated in both apo- and 2FCb-CexCD. Detailed discussions of this apparent conflict will be provided in Section 4.4.3. The apparent neutrality of Asp277 also contradicts a previous publication in which it was assumed to form a buried ion-pair with Lys302 (Poon et al, Chapter 4 - Electrostatic Interactions and Catalysis by Cex 128 Q O X © O 6 Q. 03 Q o X <D O A o LL CM Glu127^^,Glu233 • CD • rrrn Downfield Upfield Asp14 Asp123 ooooaro o o o Asp277 •Glu hGIn h Asp h Asn Glu127^ • a n r m GIU233" • f Asp14 Asp123 OOOOCOXD o o o Asp277 hGlu hGIn hAsp • Asn 186 184 182 180 178 176 174 172 1 3 C (PPm) Figure 4.13. Distribution of 1 3 c 8 / T chemical shifts in apo- and 2FCb-CexCD. The distribution of sidechain 1 3 C carboxyl chemical shifts for Glu (•), Gin (•), Asp (o), and Asn (•) residues in (top) apo- and (bottom) 2FCb-CexCD at pH 6.5 and 40 °C. n Glu233 in 2FCb-CexCD esterified with the inhibitor. Only selected residues are labelled. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 129 2006). This will be further discussed in Section 4.4.1. With respect to the assigned Glu residues, surprisingly, the observed 1 3 C 5 chemical shift of Glul27 is 182.5 ppm in apo-CexCD and 183.0 ppm in 2FCb-CexCD, suggestive of a negatively-charged sidechain in both states. This cursory interpretation is incompatible with previously-held assumptions regarding the Cex catalytic mechanism (MacLeod et al, 1996), which points to Glu 127 being a general acid (neutral) to assist the departure of the aglycone, and a general base (negatively-charged) to deprotonate a water molecule in the hydrolysis of the glycosyl-enzyme intermediate. Although Glu 127 in apo-CexCD does indeed have a more upfield chemical shift (182.5 ppm) compared to its 2FCb-CexCD form (183.0 ppm), this small change is not sufficient to confidently predict a shift in its protonation equilibrium. As will be subsequently demonstrated using amide-monitored pH titrations (Section 4.3.3.2.4.1), it was concluded that Glu 127 is in fact neutral in the free and negatively-charged in the covalently-inhibited forms of CexCD. Effects beyond its own charge state may be influencing the Glul27 1 3 C 5 chemical shift, but the available structural information does not reveal any obvious reasons for this behaviour (White et al, 1994). A summary of the Glu and Asp chemical shifts and their tentatively-derived charge states are shown in Tables 4.6 and 4.7. 4.3.3.2.4 'H- 1 5 N monitored titrations The pKa values of amino acid sidechains are best elucidated from chemical shift measurements of the 1 5 N or 1 3 C ionizable moieties as a function of pH. Although successful for the histidine residues in CexCD, the direct monitoring of the carboxyl shifts of the Asp and Glu sidechains as a function of pH was not possible. Thus, an alternative approach was to monitor the pH-dependent chemical shifts of the 'H- 1 5N pairs found in backbone and sidechain amides and the tryptophan indoles and use these as reporters of ionizable groups of interest. Amide and indole NH groups are very sensitive to changing structural and electrostatic environments due to their high bond polarizability (relative to C-H bonds) (De Dios et al, 1993) and their ability to form hydrogen bonds leading to secondary structural elements. Thus, changes to the 'H Chapter 4 - Electrostatic Interactions and Catalysis by Cex 130 Apo-CexCD 2FCb-CexCD Glu 1 3 C 5 Chemical Possible Charge 1 3 C 8 Chemical Possible Charge Residue Shift (ppm) State at pH 6.5* Shift (ppm) State at pH 6.5* 6 183.8 - 183.7 -26 184.3 - 184.4 -36 182.5 - 182.6 -43 n/a 182.4 -52 n/a# n/a* 76 183.3 - 183.2 -101 182.8 - 182.8 -116 182.4 - 182.3 -127 182.5 . t 183.0 -151 182.4 - 182.4 174 183.9 •- 183.8 -233 182.6 - 174.5 neutral 289 182.7 - 182.4 -310 183.3 - 183.3 Table 4.6. 1 3 C S chemical shifts and possible charge states of the glutamic acid residues in apo-and 2FCb-CexCD. A compilation of the 1 3 C 5 chemical shifts of the glutamic acid residues in apo-and 2FCb-CexCD at pH 6.5 and 40 °C as determined using the C 5 / Y(C rC pC aC')NH experiment. The proposed ionization states of the sidechains (*) are based solely on the observed 1 3 C 5 chemical shifts. All of the unassigned 1 3 C S resonances (n/a) are due to the unavailability of the amide assignments of the proceeding residue, with prolines specially denoted (*). (T) Based on its 1 3 C 8 chemical shift, Glul27 appears to be negatively-charged in apo-CexCD. However, based on pH-dependent chemical shifts as shown in Section 4.3.3.2.4.1, Glul27 is hypothesized to be protonated and neutral in the free form of CexCD. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 131 Apo-CexCD 2FCb-CexCD Asp 1 3 C 5 Chemical Possible Charge 1 3 C 5 Chemical Possible Charge Residue Shift (ppm) State at pH 6.5* Shift (ppm) State at pH 6.5* 9 178.6 - 178.7 -.14 177.3 titrating 177.3 titrating 20 n/a* n/a* 34 178.8 - 178.8 -49 179.2 - 179.2 -64 o/l o/l 72 179.1 - 179.2 -90 179.4 - 179.4 -113 179.1 - 179.2 -123 174.9 neutral 174.9 neutral 131 n/a n/a 138 180.6 - 180.7 -161 n/a* n/a* 170 180.6 - 180.5 -185 179.0 - 179.0 -189 180.2 - 180.2 -198 178.6 - 178.7 -214 180.5 - 180.6 -224 179.2 - 179.2 -228 178.8 - 178.8 -235 n/a 181.0 -243 n/a n/a 253 n/a n/af 277 175.9 neutral 175.9 neutral 284 179.8 - 179.8 -296 n/a n/a Table 4.7. 1 3 C r chemical shifts and possible charge states of the glutamic acid residues in apo-and 2FCb-CexCD. A compilation of the 1 3 C r chemical shifts of the aspartic acid residues in apo- and 2FCb-CexCD at pH 6.5 and 40 °C is shown. The proposed ionization states of the sidechains (*) are based solely on the observed 1 3 C Y chemical shifts as determined using the C 5 / Y(CYC pC aC')NH experiment. All of the unassigned 1 3 C 8 resonances (n/a) are due to the unavailability of the amide assignments of the proceeding (i+J) residue, with prolines specially denoted (*). (o/l) The 1 3 C Y resonance of Asp64 cannot be resolved as it is overlapped with the C resonance from Gln250. (T) The 1 3 C Y crosspeak from Asp235 in 2FCb-CexCD cannot be observed due to the extremely weak Tyr254 amide peak. Aspl4 is titrating, therefore exhibiting intermediate 1 3 C Y chemical shifts in between the upfield and downfield cluster of peaks, therefore indicative of protonated and deprotonated Asp, respectively. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 132 and 1 5 N chemical shifts as a function of pH reflect the effects of direct hydrogen bonds or indirect electric field interactions with titrating groups, as well as pH-induced conformational changes (Chen et al, 2000; Forman-Kay et al, 1992; Russell and Fersht, 1987). Furthermore, due to structural and electrostatic effects, the magnitude of the chemical shifts observed has no clear correlation to the spatial separation between the NH and ionizable groups. This is exemplified in the N-terminal domain of rat CD2, where the chemical shift of Arg31 'H e changed 0.2 and 0.5 ppm upon ionization of Glu41 and Glu29, despite residing 4.0 and 4.5 A, away respectively (Chen et al, 2000). Conveniently, the amide of an Asp residue can weakly detect the titration of its own carboxylate group via through-bond inductive effects (Richarz and Wuthrich, 1978). Unfortunately, the amide of a Glu residue rarely "senses" its intra-residue titration unless its sidechain carboxyl group interact directly with its amide (Bundi and Wuthrich, 1979). The overall result is that the pH-dependent chemical shifts of amide groups can exhibit complex titration patterns. Therefore, the availability of a high resolution structure is imperative in the interpretation and analysis of these observed changes to obtain pKa values. Of course, the corroboration of these indirect interactions using other independently determined titrations (e.g. 1 3C 5(C r)H y for Glu residues) can be useful in dissecting the observed complex titration curves derived from 'H-^N correlations alone (Chen et al, 2000). To dissect the complex electrostatic interactions in the CexCD active site, uniformly ^re-labelled apo- and 2FCb-CexCD were titrated from pH 3.5 to 9.3 and 3.6 to 9.3, respectively. In both cases, the "H-15N TROSY spectra were reassigned at each pH by tracing the "movements" of the crosspeaks, which are in fast exchange. With a large number of 'H- 1 5 N mainchain amides, sidechain indoles, and Gln/Asn amide resonances, analysis of the entire titration series for the two forms of CexCD is demanding. Compounding this effort is the presence of overlapping and/or broadening peaks, which prevent unambiguous assignments of all resonances. Therefore, for this discussion, the primary focus is on those amides and indoles which provided insight into the hydrogen bonding and electrostatic interactions found in the active site of apo- and 2FCb-CexCD. In addition, other Chapter 4 - Electrostatic Interactions and Catalysis by Cex 133 interesting nuclei which display perturbed pKa values will also be discussed. In support of conclusions drawn by using 'H- 1 5 N monitored experiments, corroborative evidence from other aforementioned lysine and histidine titrations will also be considered. 4.3.3.2.4.1 Catalytic Nucleophile (Glu233) and the Catalytic General Acid/Base (Glu 127) Using available kinetic information, it has been hypothesized that the apparent pKa value of 4.1 from the acidic limb of the kcat/Km vs. pH activity profile corresponds to Glu233, whereas the apparent pKa value of 7.7 from the basic limb corresponds to Glul27 (Tull and Withers, 1994). One indisputable fact is that when CexCD is trapped in its covalently-bound intermediate form using 2F-DNPC, the formation of the glycosyl-enzyme covalent bond renders Glu233 non-titratable (White et al., 1996). In examining the Cex active site, a large number of coupled nuclei are potentially available as reporter groups for the pH titrations of Glu233 and Glul27. Namely, Glu233 is in close proximity to the N s of Asnl69, N E of Gln203, C E l of His80, N c 2 of His205, N E l of Trp273, and N E | of Trp84, whereas Glul27 hydrogen bonds to both the sidechain N E of Gln203 and N 6 1 of Trp84 and is close to the N 8 of Asnl26. Unfortunately, the lack of NH 2 group assignments for these asparagine and glutamine residues precludes their use as convenient reporters for the pKa values of Glu233 and Glul27. In apo-CexCD, the pH-dependent 'H E l chemical shift curve for Trp273 shows a biphasic titration (Figures 4.14 and 4.15). The ionization with a pKa of 7.6 ±0.1 is attributed to Asp235 (to be shown later), and the one with pKa ~ 4.0 ± 0.2 is tentatively assigned to Glu233. This conclusion is supported by considering the His80 C E l titration curve (Figure 4.15). In CexCD, His80 is close to both Glu233 and Aspl23. With Aspl23 likely being neutral based on its 1 3 C Y shifts (Table 4.7), the pKa of 4.8 + 0.3 "sensed" by His80 must therefore belong to Glu233 (its own titration occurs at pKa 7.9). Consistent with this pKa value, Glu233 has a l 3 C 8 shift diagnostic of a deprotonated carboxylate at pH Chapter 4 - Electrostatic Interactions and Catalysis by Cex 134 Figure 4.14. 'H-1 5N NMR reporter groups used to probe for the pH titrations of Glu233 and Glul27 in apo- and 2FCb-CexCD. The (top) ball-and-stick and (bottom) schematic representation of the 'H- 1 5N NMR reporter groups (pH 6.5) used to probe for the pH titrations of Glu233 and Glu 127 in (left) apo- and (right) 2FCb-CexCD. The inter-atomic distances between reporting nuclei are shown in (A). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 135 apo-CexCD 2FCb-CexCD 139.2 • 138.8 •138.4 pKa = 7.6±0.1 (Asp235) Trp273-1HEl pKa = 7.9±0.1 (His80) pKa = 4.8±0.3 (Glu233) His80-13Cs1 = 7.5 ±0.2 (Glu127) pKa = 4.4±0.1 (Glu233) Trp84-1Hs1 10.0 PH * • * . * *—• • * pKa = 3.7±0.1 (Asp235) Trp273-1H61 His80-13Cs1 pKa = 4.2 ±0.1 (Glu127) Trp84-1HE1 4.0 6.0 8.0 10.78 10.74 10.72 10.70 10.68 10.66 •139.5 -139.0 -138.5 -138.0 -10.8 -10.7 -10.6 -10.5 -10.4 -10.3 -10.2 10.0 Figure 4.15. pH-dependent chemical shift changes of Trp273 1 H E l , His80 1 3 C E l , and Trp84 'IT1 in apo- and 2FCb-CexCD. The plots of the pH-dependent chemical shift changes of Trp273 'H E l , His80 1 3 C E l , and Trp84 'H E l in (left) apo- and (right) 2FCb-CexCD are shown. The fit pKa values and graphs to which the ionizations are attributed are shown. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 136 6.5 (Table 4.6). Upon covalent modification, the 1 3 C 5 chemical shift of Glu233 became 174.5 ppm, indicative of its presence in a glycosidic linkage (Table 4.6). Furthermore, His80 1 3 C E l in 2FCb-CexCD no longer showed any titration. Interestingly, Trp273 'H E l now titrates with a pKa of 3.7 ± 0.1, and shifts upfield rather than downfield as seen with apo-CexCD. It will be argued in Section 4.3.3.2.4.3 that this corresponds to the titration of Asp235 (i.e. whose pKa has dropped from 7.6 upon inhibition), and not the covalently-bound nucleophile. Glul27 is within hydrogen bonding distance of Trp84 N E l H. The pH-dependent chemical shifts of this indole report pKa values of 7.5 ± 0.2 and 4.4 ± 0 . 1 in apo-CexCD (Figures 4.14 and 4.15) . These are attributed to Glul27 (2.8 A away) and Glu233 (7.7 A away), respectively. Upon formation of the intermediate, Trp84 reports only one pKa at 4.2 ± 0 . 1 . Since Glu233 can no longer titrate, this ionization is assigned to Glul27. The pKa assignment of Glul27 is further supported by the chemical shifts of Tyrl71-NH, which interacts with Aspl70-O81 and G1U127-Oe2 atoms (Figure 4.16) . For Tyrl71 1 5 N in apo-CexCD, two ionizations are present with pKa values of 3.9 ± 0.3 and 7.3 ± 0.2, while only a single pKa of 3.8 ± 0.2 occurs in 2FCb-CexCD. These results are consistent with the pKa of 3.9 belonging to Aspl70 C y (negatively-charged at pH 6.5, Table 4.7), and the pKa of 7.3 belonging to Glul27 C 5 (neutral at pH 6.5, Table 4.6). Upon glycosyl-enzyme intermediate formation, Glu 127 becomes deprotonated, leading to the disappearance of the basic ionization detected by Tyrl71-15N. The resultant pKa of 3.8 must therefore reflect a combination of both the negatively-charged Aspl70-Cy and Glul27-C8 (Tables 4.6 and 4.7). These series of discernible electrostatic interactions suggest that the pKa of Glul27 "cycles" in order to fulfill its role as both a protonated (neutral) general acid and a deprotonated (negatively-charged) general base. In summary (Table 4.8), after evaluating all the available titration curves, the apparent pKa value of Glu233 is 4.4, while for Glul27, the pKa values are 7.4 and 4.2 when Cex is in its apo- and 2FCb-CexCD forms, respectively. The simple interpretation of the ionization states for Glul27 based on its 1 3 C 5 chemical shifts (Figure 4.13) appears to be erroneous, in light of the wealth Chapter 4 - Electrostatic Interactions and Catalysis by Cex 137 Asp170 Asp170 2FCb Aspno H N -Glu233 2FCb 117.7 | _ 117.6 Q . 117.5 apo-CexCD pKa = 3.9±0.3 (Asp170) TyrmlV pKa = 7.3±0.2 (Glu127) 4.0 6.0 8.0 10.0 PH 2FCb-CexCD TyrmlW1 pKa = 3.8±0.2 (Glu127 + Asp170) 4.0 6.0 8.0 118.5 118.4 118.3 10.0 Figure 4.16. pH-dependent chemical shift changes of Tyrl71 1 5 N H in apo- and 2FCb-CexCD to probe for the pH titrations of Glul27 and Aspl70. The (top) ball-and-stick and (middle) schematic representation of the 'H- 1 5N NMR reporter group (pH 6.5) used to probe for the pH titrations of Glu 127 and Asp 170 in (left) apo- and (right) 2FCb-CexCD. The inter-atomic distances between reporting nuclei are shown in (A), (bottom) The plots of the pH-dependent chemical shift changes of Tyrl71 1 5 N H in (left) apo- and (right) 2FCb-CexCD are shown. The fit pKa values and graphs to which the ionizations are attributed are shown. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 138 Residue Form Possible Charge State at pH 6.5 pKa Glu43 apo negative 3.8 2FCb negative 4.3 Glul27 apo neutral 7.4 2FCb negative 4.2 Glu233 apo negative 4.4 2FCb covalently-modified -Table 4.8. Summary of the charge states and pKa values of the Glu residues in apo- and 2FCb-CexCD. The possible charge states of the Glu residues were determined from the observed pKa values and/or 1 3 C 8 chemical shifts (Table 4.6). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 139 of amide-monitored pH titration data. The complex interplay between these differentially-charged residues and the rest of the Cex active site will be discussed later (Section 4.4). These observations demonstrate that the phenomenon of "pKa cycling" indeed does exist in another family of glycoside hydrolases beside that of the family 11 xylanase Bex (Mcintosh et al, 1996). 4.3.3.2.4.2 A substrate-binding active-site glutamate (Glu43) Glu43 resides in the CexCD active site, with a primary role of hydrogen bonding to the 2'-OH of the bound substrate via its carboxylate 0 s 2 to form a part of the (-2) binding subsite (White et al, 1996). The sole NMR reporter group for this carboxylate is its own backbone amide. Although far removed (6.9 A), this amide may electrostatically interact with the H e 2 of His80. The pH-dependent chemical shift curves of Glu43 H N for both apo- and 2FCb-CexCD are shown in Figure 4.17. When free of the substrate, Glu43 H N "senses" two ionizations, one with a pKa of 3.8 ± 0.2 that likely arises from its own sidechain and one with a pKa of 7.4 ±0.1 that correlates to the independently-determined pKa of 7.9 from His80 (see Section 4.3.2.2). The difference in these values may reflect the errors in fitting the chemical shift changes of Glu43 H N in the absence of a low pH baseline value. When covalently inhibited, the pKa of His80 decreases to less than 2.8, and hence the disappearance of this particular ionization in the curve for Glu43 H N ; the remaining ionization at pKa 4.3 ± 0.2 can be attributed to Glu43. In support of this conclusion, the downfield 1 3 C 5 chemical shift of Glu43 in 2FCb-CexCD at pH 6.5 is consistent for a deprotonated carboxyl group (Table 4.6), thereby allowing it to effectively accept a hydrogen bond from the distal 2'-OH of the bound 2F-cellobiosyl inhibitor. A summary of the obtained pKa values of Glu43 is provided in Table 4.8. 4.3.3.2.4.3 An ionization modulator (Asp235) Asp235 resides near the CexCD active site, and participates in an extended hydrogen bonding network involving Glu233, His205, and Trp281. Asp235 hydrogen bonds to His205 H 8 1 via its O 5 1 and to Trp281 H E l via its O 8 2 : The carboxylate O 8 2 is also in close proximity to Trp273-Hel (Figure Chapter 4 - Electrostatic Interactions and Catalysis by Cex 140 Figure 4.17. pH-dependent chemical shift changes of Glu43 Ur in apo- and 2FCb-CexCD to probe for the pH titrations of Glu43. The (top) ball-and-stick and (middle) schematic representation of the ' H - ^ N N M R reporter groups (pH 6.5) used to probe for the pH titrations of Glu43 in (left) apo-and (right) 2FCb-CexCD. The inter-atomic distances between reporting nuclei are shown in (A), (bottom) The plots of the pH-dependent chemical shift changes of Glu43 I H N in (left) apo- and (right) 2FCb-CexCD are shown. The fit pKa values and graphs to which the ionizations are attributed are shown. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 141 4.18) . Being its closest neighbour, the positively-charged non-titrating His205 (Figure 4.8 and Figure 4.10) displays a 1 3 C E l titration curve containing a minor monophasic transition consistent with the pKa of Asp235 being 7.9 ± 0.1 and 3.6 ± 0.3 (Table 4.7) in its apo- and 2FCb-CexCD states, respectively (Figure 4.19). The latter value cannot reflect the pKa value of the other hydrogen bonding partner to His205, Glu233, as it is non-titratable in 2FCb-CexCD. When compared to peptides in random coils, the abnormally high pKa value of Asp235 in its apo-state is further supported by the pH-dependent chemical shift curves from its other hydrogen bonding partner, Trp281-Hel ('H observed, pKa 7.7 ± 0.1) and its neighbour, Trp273-Hel ('H observed, pKa 7.6 ± 0.1) (Figure 4.19). The neutral and protonated Asp235 at pH 6.5 becomes deprotonated and negatively-charged upon covalent modification of CexCD, as demonstrated by a minor titration, extracted from the His205 C E l titration, to yield a pKa value of 3.6 ± 0.3. When examining the titration curves for both Trp281-HEl and Trp273-HEl in 2FCb-CexCD, the basic ionization is replaced by two approximately matching transitions, with pKa values of 4.4 ±0 .1 and 3.7 ± 0.1, respectively (Figure 4.19) . Although 1 3 C Y chemical shifts support the assigned pKa values of Asp235 in 2FCb-CexCD (Table 4.7), the differences observed from the expected values based on the pKa values can be attributed to errors in fitting, as well as to structural and electrostatic effects subtly affecting the chemical shifts of the reporter groups. A summary of the obtained pKa values for the various Asp residues is located in Table 4.9. The overall consequence of the "cycling" of the Asp235 pKa will be highlighted later (Section 4.4.4). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 142 Figure 4.18. H - T f NMR reporter groups used to probe for the pH titrations of Asp235 in apo-and 2FCb-CexCD. The (top) ball-and-stick and (bottom) schematic representation of the ' H - 5 N N M R reporter groups (pH 6.5) used to probe for the pH titrations of Asp235 in (left) apo- and (right) 2FCb-CexCD. The inter-atomic distances between reporting nuclei are shown in (A). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 143 138.5 138.4 Q_ 138.3 Q . apo-CexCD 2FCb-CexCD O C O 138.2 138.1 138.0 12.1 12.0 E X '"- 11.8 11.7 9.70 9.68 966 CL 9-64 CL s — 9.62 ^ 9.60 9.58 9.56 His205-13Cs • • /• pKa = 7.9±0.1 « (Asp235) J Trp281-1HE1 pKa = 7.7 ±0.1 (Asp235) ,pKa = 4.0±0.2 (Glu233) pKa = 7.6±0.1 (Asp235) Trp273-1HB1 His205-13CE • • • pKa = 3.6±0.3 (Asp235) Trp281-1HB1 pKa = 4.4 ±0.1 (Asp235) • • * . » • — • • » 'pKa = 3.7±0.1 (Asp235) 136.6 136.5 136.4 136.3 136.2 M36.1 12.30 Trp273-1HE1 4.0 6.0 8.0 10.0 4.0 PH 6.0 8.0 10.0 12.32 12.34 12.36 12.38 12.40 12.76 12.74 12.72 12.70 12.68 12.66 Figure 4.19. pH-dependent chemical shift changes of His205 1 3 C £ \ Trp281 ' H N , and Trp273 ' H E in apo- and 2FCb-CexCD. The plots of the pH-dependent chemical shift changes of His205 1 3 C s l , Trp281 ' H N , and Trp273 'H 6 1 in (left) apo- and (right) 2FCb-CexCD are shown. The fit pKa values and graphs to which the ionizations are attributed are shown. I T T E I Chapter 4 - Electrostatic Interactions and Catalysis by Cex 144 Residue Form Possible Charge State at pH6.5 pKa Aspl23 apo neutral >6.5 2FCb neutral >6.5 Aspl70 apo negative 3.9 2FCb negative n.d. Asp235 apo neutral 7.7 2FCb negative 3.9 Asp277 apo neutral ' n.d. 2FCb neutral n.d. Table 4.9. Summary of the charge states and pKa values of the Glu residues in apo- and 2FCb-CexCD. The possible charge states of the Asp residues were determined from the observed pKa values and/or 1 3 C y chemical shifts (Table 4.7). The pKa values not determined are noted (n.d.). Chapter 4 - Electrostatic Interactions and Catalysis by Cex 145 4.4 DISCUSSION 4.4.1 Lysines The protonation states of several lysines in CexCD under near-physiological conditions were successfully investigated and their ionization states unambiguously determined. Lys302 in both apo-and 2FCb-CexCD, and Lys47 in the 2FCb-inhibited form, were detected through 'H- 1 5 N HMQC NMR experiments at 30 °C and pH 6.5. These readily observed amines must therefore be significantly protected from HX exchange. The amine of Lys302 forms hydrogen bonds with the carbonyl oxygens of Ala291 and Leu293, and to the carboxyl of Asp277 (see 4.3.3.2.4.3 and Figure 4.18). The latter was previously assumed to form an ion-pair with Lys302 (Pbon et al, 2006). However, its distinct upfield-shifted 1 3 C y resonance suggests that Asp277 is actually neutral at pH 6.5. There exists the possibility that the Asp277 is negatively-charged as strong "ionic" hydrogen bonds would potentially result in the typically downfield resonance being shifted upfield due to the acceptance of the proton from the hydrogen bond donor (Warshel et al, 2006). Attempts to measure the pKa value of this aspartic acid through the use of reporter ] H- 1 5 N groups were unsuccessful. The Asp277 O 8 1 is 2.8 A from Leu293 NH, whereas the Asp277 O 8 2 is 3.4 A away from Asp277 NH. Unfortunately, spectral overlap prevented the extraction of meaningful pKa values from the ' H N and 1 5 N H shifts of these residues. Overall, it appears that the unfavourably buried positively-charged Lys302-NH3+ group is stabilized via a constellation of weak charge-dipole interactions with Asp277, Ala291 and Leu293, which in summation, may be equivalent to the total stabilization offered by a buried ion-pair. These arrays of interactions also account for its protection from exchange (Figure 4.20). The formation of the stable glycosyl-enzyme intermediate, upon reaction with 2F-DNPC as revealed by X-ray crystallography, resulted in Lys47 being hydrogen bonded to both the distal and proximal sugar moieties of the cellobioside, thereby leading to its burial Chapter 4 - Electrostatic Interactions and Catalysis by Cex 146 Figure 4.20. Stabilization of the positively-charged Lys302. The ball-and-stick (left) and schematic (right) representations of the stabilization of the positively-charged Lys302 by the Asp277 carboxylic acid, and the backbone carbonyl oxygens of Ala291 and Leu293 at pH 6.5. The inter-atomic dipole-charge interactions are highlighted, with their respective Lys302-N^ to oxygen distances denoted. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 147 and protection from H X (Figure 4.2). The structural information further supports the observed data from the ^-coupled ' H - ^ N HSQC experiments and pH titrations, which reported that Lys47 must be a hydrogen bond donor while in its protonated (-NH3+) state at pH 6.5. The importance of these interactions is highlighted by a 75-fold decrease in kc a t /K m of the Cex K47A mutant for hydrolysis of 2,4-dinitrophenyl P-cellobioside relative to the wild-type enzyme (Wicki et al, 2007a). Since there are 18 lysines present in this system, it is clear that many undergo rapid hydrogen exchange with water, thereby precluding their detection. This includes the exposed Lys47 in the active site of apo-CexCD. Use of strategies to slow the exchange with water (decreasing temperature and pH) was successful in extracting the signals for up to 10 of the amines, with potentially more resonances since many may be overlapped in the spectrum. Unfortunately via spectral comparison, it was not possible to identity a peak corresponding to Lys47 in apo-CexCD, and this was attributed to the unfavourable relaxation properties present for this specific lysine. That is, the full side chain 1 3 C spin systems of surface lysines, but not the internal Lys47, were assignable in the C(C')TOCSY-NH spectra (Cavanagh et al, 2006) of 70% 2H/13C/15N-labelled CexCD (Appendix 1 and Appendix 2). Regardless, due to the absence of the detectable amine signal, one cannot determine the ionization state of Lys47 in apo-CexCD. However, it is logical to assume that it is also in its protonated - N H 3 + form since it will be reasonably solvent-exposed. 4.4.2 Non-active site histidines (His 85, Hisl07, Hisll4) In addition to catalytically important histidines, NMR measurements also yielded information on other "structural" histidines in CexCD. His85 lies partly near the surface of the Cex active site, in close proximity to the guanidinium group of Argl36, as well as the carboxyl of the general acid/base catalyst Glul27. His85 has a pKa value of < 2.8 and remains in its neutral N 6 2 tautomer under all pH conditions examined. The decrease in the pKa of His85 appears to be primarily driven by its Chapter 4 - Electrostatic Interactions and Catalysis by Cex 148 neighbouring positively-charged guanidinium group, while the cycling of the pKa of Glul27 does not appear to play a significant role on the charge of the His85 imidazole ring in both apo- and 2FCb-CexCD. The positively-charged His 107 is located within the interior of CexCD, forming a salt bridge with the presumably negatively-charged Glu52 (unfortunately its 1 3 C 5 chemical shift was not assignable) as well as a hydrogen bond to an internal water. These interactions have the effect of elevating the pK a of His 107 to > 10.4. His 114 is exposed on the surface of CexCD, and donates hydrogen bonds to a bound water molecule seen in the crystal structure and to the negatively-charged Asp64 carboxyl group (Table 4.7). Unlike the previously-mentioned charge pair of Hisl07-Glu52, which resides in the low dielectric interior of CexCD, His 114 is solvent-exposed. This leads to the pKa value of 8.1, which is only -1.5 units higher than expected for a histidine in a random-coil polypeptide (Creighton 1993). 4.4.3 Interactions between Aspl23, HisSO, and Glu233 The hypothesized interactions between Asp 123, His80, and Glu233 are summarized in Figure 4.21. In both apo- and 2FCb-CexCD, Asp 123 appears'to be protonated and neutral, accepting a hydrogen bond from His80 N 8 1 H. This conclusion is based on its upfield 1 3 C V shift, indicative of an aspartic acid. Unfortunately, its pKa value was not determined due to the lack of 'H- 1 5 N reporter groups nearby. The neutrality of Asp 123 may simply be a result of its exclusion from solvent in a low-dielectric environment. His80 switches from being positively-charged (pKa = 7.9) to being neutral in its N 8 1 tautomeric state (pKa < 2.8) upon covalent modification of CexCD. This dramatic decrease in pKa appears to be driven by three factors. Firstly, upon binding of the inhibitor, His80 becomes significantly more buried, thereby favouring its neutral form by virtue of solvent exclusion. Secondly, while in its N 8 1 tautomeric form, it can also accept a stabilizing hydrogen bond from the 3-OH of the bound inhibitor, which in turn accepts a hydrogen bond from Lys47. An additional benefit of being in this tautomer is that His80 can continue to donate a hydrogen bond to the neutral Asp 123. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 149 D123 Figure 4.21. Inter-residue interactions between Aspl23, His80, and Glu233. The ball-and-stick (top) and the schematic (bottom) representations o f the highlighted inter-residue interactions and distances (A) between A s p l 2 3 , His80, and Glu233 at p H 6.5 in the free (left) and the covalently-inhibited (right) C e x C D . Chapter 4 - Electrostatic Interactions and Catalysis by Cex 150 Finally, the loss of a charge-charge interaction with the now neutral Glu233 (apo pKa = 4.3) would also tend to decrease the pKa of the nearby His80 (4.0 A). These observations are refinements to previous X-ray crystallographic analysis of inhibited Cex and other related Family 10 glycosyl hydrolases, which assumed that either the ionized histidine or its opposite N 6 2 tautomer would be donating a hydrogen bond to the 3-0 of the substrate (Gloster et al, 2003; Notenboom et al, 2000; Varrot et al, 2003; White et al, 1996). In apo-CexCD, the elevated pKa of His80 (7.9) means it is positively-charged at pH 6.5. Any potentially detrimental charge-charge repulsion with the positively-charged neighbouring Lys47 amine (pKa > 9) (4.5 A) would be reduced by the intervening solvent. The close association between His80 and Glu233 is further highlighted in the kinetic study of the CexCD deletion mutant, H80A by extracting the observed apparent pKa values (Figure 4.22). This mutant exhibits a ~ 2000 fold decrease in k c a t / K m from 1500 m M ' V for WT-Cex to 0.72 m M ' V towards the synthetic substrate PNPX2 (j>H 7, 37 °C). Furthermore, the bell-shaped activity profile of the free H80A-CexCD enzyme has an apparent acidic and basic limb pK a of 4.6 and 6.2, respectively (c.f. to 4.1 and 7.7 in WT-Cex towards the synthetic substrate PNPC). Since the acidic limb pK a is assigned to be the Glu233 nucleophile, this increase in apparent pK a may have arisen from the removal of a favourable electrostatic interaction with the nearby His80 (His80-Ne2 to Glu233-0El separation 4.0 A). The basic limb of the pH dependence of k c a t / K m is assigned to the general acid/base residue, Glul27. With the His80 N e 2 located 7.1 A away from the neutral Glul27 O e 2 , any hypothetical electrostatic interactions between these two residues should also result in the increase in the pKa of Glu 127 once the positive His80 is removed. Instead, the apparent basic pKa decreased. This emphasizes further that the analysis of apparent pKa values from kinetic studies can be very tricky, as the observed changes can be a complex combination of structural and electrostatic perturbations. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 151 pH Figure 4.22. pH-dependence of wild-type and H80A Cex towards PNPX2. Mutation of His80 to alanine changes the pH-dependence of kcat/Km. The activity profile of full length H80A Cex towards > PNPX2 (•) is fit to a bell-shaped curve with apparent pKa values of 4.6 and 6.6. The activity of wild type Cex towards PNPC (o) follows pKa values of 4.1 and 7.7 (reproduced from Tull and Withers, 1994). Note that, with wild type Cex, kca t / K m is ~ 100 fold higher towards PNPX2 versus PNPC (Notenboom et ai, 1998). Also, the pH-dependence of k c a t / K m reflects ionization events within the free enzyme and thus should not differ between the two closely related neutral substrates. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 152 4.4.4 Interactions between Glu233, His205, and Asp235 The catalytic nucleophile, Glu233, is absolutely essential in the Cex mechanism. Its 0 E ionization, and hence its pKa, is modulated by a host of active site residues, including the previously mentioned His80 and Glul27. Moreover, it associates most closely with the continuously positively-charged His205 (pKa > 10.4 and > 10.1 for apo- and 2FCb-CexCD, respectively) via a hydrogen bond to its N e 2 H. In apo-CexCD, kinetic measurements suggest that Glu233 is negatively-charged at neutral pH, with an apparent pK a value of ~ 4.1 in the free enzyme (MacLeod et al, 1994). The available 1 3 C 5 chemical shifts (Table 4.6) are consistent with it being deprotonated at pH 6.5, and pH titrations using . various amide and indole reporter groups (Figures 4.15) support the conclusion that its apparent pKa value is 4.2. In apo-CexCD, the protonated His205 stabilizes both the negatively-charged nucleophile and the neutral Asp235 (Figure 4.23). This arrangement of Asp235-His205-Glu233 is strikingly similar to those of the catalytic triads (nucleophile - His - Asp/Glu) found in some serine proteases (Bachovchin, 2001), phosphatidylinositol-specific phospholipase C (Ryan et al, 2001), and cholinesterases (Massiah et al., 2001; Viragh et al., 2000). Interestingly, Asp235 remains protonated and neutral in apo-CexCD at pH 6.5, possessing a pKa value of 7.7. This is most likely driven by its burial from the aqueous solvent. When Cex is inhibited, the loss of the titratable behaviour from Glu233 is expected due to the formation of the glycosyl-enzyme covalent bond. His205 remains protonated and positively-charged, such that it can support a bifurcated hydrogen bond from its N e 2 H to both Glu233-Oel and the 05 of the proximal sugar ring (3.0 A) (White et al, 1996). In turn, the pKa of Asp235 decreases to 3.9 to form a buried ion-pair with His205. This suggests that the positive charge of His205 is balanced by one negative charge (e.g. Glu233 in apo-CexCD and Asp235 in 2FCb-CexCD). The important contribution of His205 to catalysis was highlighted in a previous kinetic study, where it was discovered that the Cex H205A mutation significantly impacted Chapter 4 - Electrostatic Interactions and Catalysis by Cex 153 Figure 4.23. Inter-residue interactions between GIu233, His205, and Asp235. The ball-and stick (top) and the schematic (bottom) representations of the highlighted inter-residue interactions and distances (A) between Glu233, His205, and Asp235 at pH 6.5 in the free (left) and the covalently-inhibited (right) CexCD. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 154 both the glycosylation and deglycosylation step of the enzyme mechanism, as seen in a drop of 300-fold in kcat /K m value, and a drop of 4700-fold in k^ value, respectively, towards the substrate 2,4-DNPC. This indicates a very important role of His205 in the deglycosylation step, likely involving stabilization of the carboxylate Glu233 as it departs from the sugar anomeric centre. (Notenboom et al, 1998a) The pH-dependence of k c a t / K m is also significantly different for this H205A mutant, with only one ionization observed at a pKa value of ~ 7.5 (Notenboom et al, 1998a). Again, these changes are difficult to rationalize, but a possibility is that the Glu233 ionization is depressed outside of the examined pH range, while the Glul27 ionization dropped from 7.7 (Tull and Withers, 1994) to ~ 7.5 in the mutant. 4.4.5 Interplay between the nucleophile Glu233 and the general acid/base Glul27 The concept of "pKa cycling" was first demonstrated in a family 11 xylanase Bex (Mcintosh et al, 1996), which also undergoes a P-retaining double-displacement mechanism similar to that of Cex (Gebler et al, 1992). In a concerted manner, the active site residues in Bex modulate the pKa of the nucleophile (Glu78), such that it is negatively charged at the pH optimum. Through electrostatic repulsion, this in turn resulted in the elevation of the general acid's (Glul72) pKa value (Joshi et al, 2001). In studies where Bex is covalently-inhibited, or where the nucleophile is replaced with neutral amino acids by mutation, the measured pKa value of the general acid/base residue decreases by ~ 2.5 units from 6.7. Hence, this phenomenon of "pKa cycling" is driven predominantly by the negatively-charged nucleophile becoming neutral during the course of the reaction mechanism. Additional smaller contributions may arise from the concerted interactions with other active site residues, and from the resulting glycosyl-enzyme intermediate formation or structural perturbations (Joshi et al, 2001; Mcintosh et al, 1996) as a result of introducing mutations. In examining CexCD, the pKa of its general acid/base residue, Glul27, cycles from 7.4 to 4.2 in the free and covalently-inhibited enzyme forms, respectively. Without any other titratable groups in Chapter 4 - Electrostatic Interactions and Catalysis by Cex 155 its immediate vicinity, it is most likely that the observed ionization states of Glu 127 are set by Glu233 which is ~ 5.6 A away. While lacking neighbouring ionizable residues, the Glul27 carboxylate remains an integral part of a complex hydrogen bonding network, making it difficult to even predict the direction of pKa changes upon modification of the active site. For example, the formation of the covalently-inhibited CexCD removes the negatively-charged Glu233, and thus removes the destabilization of the conjugate base form of Glu 127, resulting in its pKa dropping back to 4.1, a more "normal" value. The partial "removal" of the negative charge on Glu233 by increasing the distance between the two groups (via mutation to an aspartic acid) might have been expected to result in a similar lowering of the pKa of Glul27. However, based on kinetic measurements, the apparent pKa of Glul27 in the Cex E233D mutant was actually elevated, thereby strongly suggesting that other electrostatic and/or structural interactions are taking place in a cooperative manner to modulate the pKa values of the active site residues (Figure 4.3) (MacLeod et al, 1996). Other similar anomalies were observed in various Cex mutants. For example, the removal of the general acid/base residue (E127A) increased the apparent pKa of Glu233 from 4.1 to ~ 6 (MacLeod et al, 1996; Tull and Withers, 1994). For complex systems such as CexCD, it is risky to distill inter-residue interactions on an individual basis in attempts to rationalize observed kinetics and mechanistic consequences of modifications made. Furthermore, kinetically derived pH-dependence relationships may reveal apparent pKa values which are the resultant of a collection of residues, rather than just from a single one. To better understand the subtle interplay between active site amino acid residues, it is therefore critical to ascertain individual pKa values using techniques capable of directly observing the ionization states of these residues. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 156 4.5 CONCLUSION < Using NMR spectroscopy, I have dissected the electrostatic interactions (including pKa values) which help to modulate the charge states of ionizable residues that take part in the enzymatic degradation of glycosides by the family 10 glycoside hydrolase Cex (Figure 4.24). Focusing on the catalytic general acid/base residue, it was demonstrated that its pKa value cycles between 7.4 and 4.2 reflecting its functional roles as a general acid and a base, respectively. As previously demonstrated, the pKa values of ionizable sidechains in Cex can be significantly perturbed upon formation of the glycosyl-enzyme intermediate. With more complex protein-protein and/or protein-substrate interactions in larger and more complicated systems, including proteins which associate with membranes, even more dramatic pKa ranges may exist for their constituent ionizable groups. As shown in this chapter, the evaluation of experimental NMR titrations can be a challenge. Thus there is a need to develop new techniques, or optimize existing ones such as neutron diffraction, to help understand the complex interplay between charged and polar residues in enzyme catalysis. Particularly humbling are the magnitudes of some of the pKa shifts observed (His pKa's ranging over at least 8 units) and the associated energies involved. Studies of this type are starting to dissect some of the possible ways in which enzymatic catalysis is affected by providing the enormous transition state stabilizations of up to 20 to 25 kcal/mol necessarily associated with the possible 1017 fold rate enhancements for catalyzed reactions. Encouragingly, quantum mechanical electrostatic calculations are beginning to be able to cope with larger systems and thus, detailed site-specific descriptions such as those described herein are particularly valuable as a benchmark for these theoretical investigations. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 157 Figure 4.24. Inter-residue and inter-molecular interactions and pKa values of the active site ionizable groups in apo- and 2FCb-CexCD. The hydrogen bonding network within the active site of (A) apo- and (B) 2FCb-CexCD are shown at pH 6.5. Using NMR-monitored pH titrations, the determined pKa values for the ionizable groups in the active site are indicated. While non-ionizable residues are shown in black, sidechains with acidic and basic pKa values are coloured in red and blue, respectively. Chapter 4 - Electrostatic Interactions and Catalysis by Cex 158 D123 W281 Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 159 Chapter 5 Probing for the -3 Glycone Binding Subsite in Cex Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 160 5.1 INTRODUCTION Catalysis by enzymes occurs rapidly and often with exquisite stereospecificity. The chemistry that takes place is dictated by the physiochemical complementarities of the active site and its substrates. However, binding of polymeric substrates is often further established by amino acid residues far removed from the catalytic residues. These extended binding clefts help dictate the specificity and affinity of natural substrate recognition for optimal catalysis. Family 10 xylanases, which adopt a fold comprising of an (ct/p^-barrel with an active site rich in aromatic and hydrophilic residues, are model systems for studying glycoside hydrolase-substrate interactions (Charnock et al, 1998; Pell et al, 2004a; Schmidt et al, 1999; White et al, 1994) (Figure 5.1). This cleft comprises of a series of subsites, each capable of binding to a xylose moiety. The subsites that bind to the glycone and the aglycone regions of a substrate are prefixed with - and +, respectively, with the numbering commencing at the site of bond cleavage (i.e. the glycosidic bond between subsites -1 and +1 is cleaved by the enzyme) (Figure 5.2) (Charnock et al, 1998; Davies et a/.,. 1997). For this family of xylanases, the two absolutely conserved catalytic glutamic acid residues act as the "boundary" between the glycone (-) and the aglycone (+) binding regions. The aglycone and the glycone sites, together, serve to bind substrates in the correct conformation for catalysis. The glycone region is often referred to as the "substrate recognition area" and the aglycone region as the "product-release area" (Schmidt et al, 1999). Xylanases hydrolyze natural xylan, which is often modified with 4-O-methyl-D-glucuronic acid residues attached at 02, or with arabinofuranose and acetyl groups at 02 and/or 03 of the xylan backbone (Xie et al, 2006). Originally, it was believed that these additional sidechains are fully cleaved by other glycoside hydrolases and esterases before the hydrolysis of the main polysaccharide chain by xylanases (Gilbert and Hazlewood, 1993). More recently, it has become clear that many xylanases can cleave partially decorated xylan chains. For example X-ray crystallography has shown how arabinofuranose sidechains can fit within the -2 binding subsite of the family 10 CmXynlOB Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 161 Figure 5.1. Cex surface binding cleft. The surface representation of the X-ray crystal structure of the Cex catalytic domain covalently bonded with the 2F-xylobiosyl inhibitor (green) is shown (Notenboom et al., 1998b). The substrate binding cleft is highlighted, showing the catalytic nucleophile and the general acid/base residues (blue), aromatic residues (yellow), and polar/charged residues (red). Figure 5.2. Subsite nomenclature. The subsite nomenclature for a generic xylanase containing 3 glycone (-) and 2 aglycone (+) binding subsites. Hydrolysis occurs in between the -1 and +1 subsites. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 163 from Cellvibrio mixtus without any identifiable enzyme-substrate interactions (Pell et al, 2004b). Arabinose sidechains of xylan bound to TaXynlOA from Thermoascus aurantiacus make both direct and solvent-mediated hydrogen bonds with the enzyme (Vardakou et al, 2005). Furthermore, these potentially bulky additions can be accommodated in both the glycone and the aglycone binding regions (Fujimoto et al, 2004; Pell et al, 2004b; Vardakou et al, 2005). It would appear to be an evolutionary advantage for organisms to produce "promiscuous" glycoside hydrolases that are able to hydrolyze a variety of substrates, rather than depending on a system of several sequentially-functioning enzymes. 5.1.1 The family 10 xylanase Cex Many studies into the mechanism and the binding specificity of the family 10 xylanase Cex have been performed. However, the vast majority of these structural and kinetics analyses utilized synthetic disaccharide-based substrates and inhibitors (Table 5.1) (MacLeod et al, 1994; MacLeod et al, 1996;McCarter et al, 1993; Notenboom et al, 1998a; Notenboom et al, 1998b; Notenboom et al 2000; Poon etal, 2007a; Schubert et al, 2007; Tull et al, 1991; Tull and Withers, 1994; Wicki et al, 2007a; Wicki et al, 2007b; White et al, 1994; White et al, 1996; Ziser et al, 1995). The available crystal structures of mutants and wild-type CexCD in the apo, noncovalently inhibited, and covalently inhibited forms clearly illustrate the binding interactions between the xylose moieties and the active site amino acid residues in the -1 and -2 subsites (Notenboom et al, 1998b; Notenboom et al, 2000; White et al, 1994; White et al, 1996). The detailed pKa analysis of these residues also helped to define the precise roles of the hydrogen bond donors and acceptors in the enzymatic mechanism (Chapter 4). Unfortunately, due to the availability of only disaccharide inhibitors, detailed binding information is not available for subsites other than at the -1 and -2 positions (see Figure 4.2 for interactions between Cex and 2-deoxy-2-fluoro-cellobioside). Studies of the cleavage patterns of xylan arid longer xylo-oligosaccharides by Cex suggested the possibility that the enzyme contains a Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 164 Substrates K m (mM) KJKm (s"1 mM"1) 2,4-Dinitrophenyl glucopyranoside3 18.0 1.9 9.5 2,4-Dinitrophenyl cellobioside" 12.9 0.11 117 /7-Nitrophenyl glucopyranosideb 0.024 8.3 0.0029 ^-Nitrophenyl cellobiosideb 15.8 0.60 26.3 /j-Nitrophenyl xylopyranosideb 2.6 20 0.13 p-Nitrophenyl xylobiosideb 39.8 0.018 2200 l%Xylanb 0.47 - -Inhibitors ki (min1) Ki(mM) kj/Kj (min 1 mM"1) 2,4-Dinitrophenyl 2-deoxy-2-fluoro-glucopyranoside0 2.5 xlO"4 4.5 5.6 xlO"5 2,4-Dinitrophenyl 2-deoxy-2-fluoro-cellobiosided 0.067 0.11 0.61 2,4-Dinitrophenyl 2-deoxy-2-fluoro-xylobioside6 0.057 0.0035 16.3 Table 5.1. Kinetic parameters for selected mono- and di-saccharide substrates and inhibitors used to probe the active site of Cex. The data were compiled from (a)(Tull and Withers, 1994), ^(Notenboom et al, 1998b), (c )(Tull et al, 1991), (d)(McCarter et al, 1993), and (e)(Ziser et al, 1995). Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 165 total of three glycone (-) and two aglycone (+) binding sites (Charnock et al, 1998). The -3 subsite was determined to provide a small, but favourable binding energy of -1 kcal/mol, which is significantly weaker than at the other Cex subsites (-2: ~ -6 kcal/mol; +2: ~ -4 kcal/mol) (Chamock et al, 1998). Contrary to these findings, an argument against a relevant -3 subsite is provided by the exclusive release of xylobiose products when Cex is incubated with xylotetraose. However, this study did not preclude the possibility that binding interactions at the +2 subsite are essential for any hydrolysis event catalyzed by Cex (Pell et al, 2004a). The precise molecular interactions involved in the putative -3 binding subsite in Cex have remained unclear as no structural information on ligands bound at this subsite was available. In an effort to quantitatively determine the contributions made by the proposed -3 site, the synthetic xylotriose-based inhibitors 2,4-dinitrophenyl 2-deoxy-2-fluoro-p-xylotrioside (2FO-DNPX3) and 2,4-dinitrophenyl 2-deoxy-2fluoro-4II-thio-P-xylotrioside (2FS-DNPX3) were used for kinetic inhibition and structural studies with CexCD (Figure 5.3). Since Cex is an endolytic xylanase, the thio-linkage in 2FS-DNPX3 should prevent its hydrolysis in between X3 and X2 as the sulfur atom has poor hydrogen bonding ability, which renders the 5,0-acetal unable to significantly benefit from general acid catalysis (Macauley et al, 2005). Based on similar inhibition kinetics as seen for 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-xylobioside (2F-DNPX2), combined with the absence of any structural contacts of the distal xylose with the active site of Cex, these data demonstrated that this enzyme does not possess a significant -3 subsite. Thus, within the limits of these measurements, glycone recognition mediated entirely by the -1 and the -2 subsites. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 166 Figure 5.3. Synthetic inhibitors of Cex. The synthetic mechanism-based inhibitors of Cex: (top) 2FO-DNPX3, (middle) 2FS-DNPX3, and (bottom) 2F-DNPX2. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 167 5.2 MATERIALS AND METHODS 5.2.1 Protein expression and purification The expression of CexCD in a synthetic medium (Muchmore et al, 1989) and its subsequent purification was performed according to previously described protocols (Chapter 2; Poon et al, 2007a). The purified protein was concentrated to a final concentration of 65 mg/mL and stored in 50 mM sodium phosphate and 0.02% sodium azide at pH 6.5. 5.2.2 Synthetic mechanism-based inhibitors The synthetic mechanism-based xylotrioside inhibitors, 2FS-DNPX3 and 2FO-DNPX3, were chemically synthesized and characterized by Terrence Kantner, and their respective structures are shown in Figure 5.3. The concentrations of these inhibitors were determined spectrophotometrically (e284 = 11110 M^cm"1) as determined using 2,4-dinitrophenyl-lactoside as a reference compound. 5.2.3 Crystallization of the CexCD-inhibitor complexes Crystals were obtained by the hanging-drop vapour diffusion method based on a previously described protocol for CexCD (Bedarkar et al, 1992). After slight optimization of the buffering conditions, the hanging drops, containing 2 uL of CexCD (65 mg/mL) and 2 pL of polyethylene glycol (PEG) 4000 in 0.1 M sodium acetate at pH 4.6, were equilibrated against 1 mL of this same buffer. After one week, large crystals were formed, and these were used to produce the CexCD-inhibitor complexes as previously described (White et al, 1996). The native CexCD crystals were soaked for 24 hours in 5 mM 2FO-DNPX3 or 2FS-DNPX3, dissolved in the above reservoir buffer. X-ray diffraction, and structural analysis and refinements were performed by Dr. Igor D'Angelo in the laboratory of Dr. Natalie Strynadka at the University of British Columbia. The atomic coordinates of the X-ray-derived structures of CexCD complexed with 2FO-DNPX3 (2FOX3 -CexCD) and 2FS-DNPX3 (2FSX3-CexCD) will be deposited in the Protein Data Bank (http://www.rcsb.org/pdb/). Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 168 5.2.4 Kinetic inactivation measurements The equilibrium binding constant (Kj) and the inactivation rate constant (kj) were measured for both 2FO-DNPX3 and 2FS-DNPX3 inhibitors according to documented protocols (Tull and Withers, 1994; Ziser et al, 1995). Samples of 2FO-DNPX3 (0, 8.8, 10.5, 11.7, 17.6, or 23.4 uM) and 2FS-DNPX3 (0, 1.2, 1.8, 3.0, 4.5, or 7.6 uM) were incubated with CexCD (0.054 mg/mL) in 1 mL mixtures containing 50 mM sodium phosphate and 1 mg/mL bovine serum albumin at pH 7.0 and 37 °C. Aliquots (10 uL) of the reaction mixtures were removed at regular time intervals and diluted into reaction cells containing 1 mL of saturating amounts (0.5 mM) of o-nitrophenyl-P-D-xylobioside (ONPX2) in the above buffering conditions at 37 °C. The residual-enzymatic activities were monitored based on dinitrophenolate release using a Varian Cary 4000 UV/Vis spectrophotometer. At each inhibitor concentration, the residual rates versus time were fit to an exponential decay to obtain the pseudo-first-order rate constant, &0bs for inactivation. Values of the equilibrium binding constant (Kj) and the inactivation rate constant (kj) were extracted by the fitting of kobS at each inhibitor concentration to Equation 5.1. k rn knh, = — L J ^ L - Equation 5.1 Kj+[I] As a control, analysis of the inactivation kinetics for 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-xylobioside (2F-DNPX2) was repeated according to Ziser et al. (Ziser et al, 1995) at inhibitor concentrations of 0.5, 1.1, 2.2, 4.4, 6.5, 10.9, or 16.3 uM. 5.3 RESULTS 5.3.1 CexCD inactivation kinetics The incubation of CexCD with the three mechanism-based inhibitors, 2F-DNPX2, 2FO-DNPX3, and 2FS-DNPX3 resulted in the time-dependent decreases in residual enzymatic activities as Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 169 shown in Figure 5.4. Pseudo-first-ordef rate constants determined at each inhibitor concentration were subsequently fit to obtain the inactivation parameters kj and Kj as summarized in Table 5.2. The 2FS-DNPX3 inhibitor exhibit comparable inhibition properties to the xylobiosyl control (kj and Kj values of 0.041 min"1 and 2.0 uM, and 0.049 min"1 and 2.4 pM, respectively). Thus a third xylose moiety does not contribute significantly to the kinetics of Cex inhibition. In comparing the inactivation data for the xylofriosyl inhibitor 2FO-DNPX3 against that of the xylobiosyl control, it was surprising that the addition of an extra distal xylose moiety resulted in worse apparent kinetic inhibition properties. That is, higher concentrations of these inhibitors were needed to obtain inactivation. Furthermore, the resultant apparent kobs could not be successfully fit to Equation 5.1, thus precluding the extraction of kj and Kj values. These observations suggested that the inhibitor was being partially hydrolyzed during the inactivation assay. Consistent with these arguments, the covalent 2FSX3-CexCD complex was detected by mass spectrometry, whereas the enzyme was not measurably labelled with 2FO-DNPX3 or with any of the potential mono- or disaccharide cleavage products (O. Hekmat, personal comm.). 5.3.2 X-ray crystal structures of inhibitor-enzyme complexes The soaking of the CexCD crystals with the inhibitors 2FO-DNPX3 or 2FS-DNPX3 yielded refined X-ray crystallographically derived structures of the corresponding covalent glycosyl-enzyme intermediates (Figures 5.5 and 5.6) with the crystal statistics shown in Table 5.3. The electron densities for the bound glycosyl rings were clearly identified for both complexes (Figure 5.7),confirming that the inhibitors reside in the active site with the anomeric centres covalently bonded to the catalytic nucleophile (Glu233) in a-conformations via ester bonds. The active site amino acid sidechains in the complexes, as well as the first two proximal xylose moieties in the xylotriose inhibitors, superimpose almost perfectly with the existing structure of the 2F-xylobiosyl-CexCD complex (Notenboom et al, 1998b) (core RMSD = 0.25 A and 0.26 A for 2FOX3-CexCD Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 170 ) Figure 5.4. Cex inactivation kinetics. The semilogarithmic plots of the residual activity of CexCD vs. time at the indicated inactivator concentrations for (A) 2F-DNPX2 ((+) 0.5 uM; (•) 1.1 uM; (o) 2.2 uM; (•) 4.4 uM; (•) 6.5 uM; (A) 10.9 pM; (T) 16.3 uM), (B) 2FS-DNPX3 ((•) 1.2 pM; (o) 1.8 uM; (•) 3.0 uM; (•) 4.5 uM; (A) 7.6 uM), and (C) 2FO-DNPX3 ((•) 1.2 pM; (a) 2.3 uM; (+) 3.5 uM; (•) 8.8 uM; (o) 10.5 uM; (•) 11.7 pM; (D) 13.5 pM; (A) 17.6 uM; (T) 23.4 uM). The extracted k o b s from A, B, and C were plotted against each inhibitor concentration used, fitted to Equation 5.1, and shown in D, E, and F, respectively. 2FO-DNPX3 is most likely hydrolyzed during the inactivation assay, and therefore, the resultant data cannot be simply fit using Equation 5.1. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 171 Inhibitors k j (min"1) K i ( u M ) k j / K j (min1 u M " 1 ) 2,4-Dinitrophenyl 2-deoxy-2-fluoro-cellobioside 0.061 120 0.00051 2,4-Dinitrophenyl 2-deoxy-2-fluoro-xylobioside 0.049 2.4 0.020 2,4-Dinitrophenyl 2-deoxy-2-fluoro-P-xylotrioside 0.041 2.0 0.021 2,4-Dinitrophenyl 4II-thio-p-xylotriosidet partially hydrolyzed partially hydrolyzed partially hydrolyzed Table 5.2. Summary of k , and K j results for CexCD. The inactivation studies of CexCD summarizing the kj, and Kj values, as well as the calculated kj/Kj values, for the various inhibitors. (t)Values obtained for 2F-0-DNPX3 cannot be quantitatively compared since the inhibitor appears to be enzymatically hydrolyzed. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 172 Figure 5.5. X-ray crystal structure of 2FOX3-CexCD. (Top) A representation of the CexCD covalently modified with 2FO-DNPX3. Active site residues that form stabilizing hydrogen bonds and van der Waal contacts to the bound inhibitor are shown. The distal X3 xylose moiety is solvent exposed and not involved in any stabilizing enzyme-ligand interactions. The sidechain oxygen atoms are coloured in red, and the nitrogens are in blue. Only the X2-X3 glycosidic oxygen is highlighted, with the remainder of the inhibitor shown in green. (Bottom) The schematic representation of the CexCD active site highlights the inter-atomic distances (A) between the enzyme-ligand hydrogen bonding partners. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 173 N126 Figure 5.6. X-ray crystal structure of 2FSX3-CexCD. (Top) A representation of CexCD covalently modified with the inhibitor 2FS-DNPX3. Active site residues that form stabilizing hydrogen bonds and van der Waals contacts to the bound inhibitor are shown. The distal X3 xylose moiety is solvent exposed and not involved in any stabilizing enzyme-ligand interactions. Note that this xylose ring is rotated ~ 180° about the glycosidic bond in comparison to the bound 2FO-DNPX3 inhibitor shown in Figure 5.5. The sidechain oxygen atoms are coloured in red, and the nitrogens in blue. Only the X2-X3 glycosidic sulfur is shown coloured in yellow whereas all other inhibitor atoms are green. (Bottom) The schematic representation of the CexCD active site highlighting the inter-atomic distances (A) between the enzyme-ligand hydrogen bonding partners. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 174 • 2FOX3-CexCD 2FSX3-CexCD Data collection X-ray source CuKa CuKa Wavelength (A) 1.541 1.541 Spacegroup P4i2,2 P4A2 Unit cell (A, deg) a=87.3, b=87.3, c=80.2 a=87.3, b=87.3, c=80.3 Resolution (A) 1.60 1.66 Highest shell (A) 1.60-1.69 1.66-1.75 # observations 469,035 (63,679) 411,044(54,950) Unique reflections 41,105 (5,850) 36,771(5,148) I/a 34.7 (8.8) 38.7(13.5) Rsym(%)* 5.2 (23.8) 4.8 (15.9) Completeness (%) 99.7 (99.2) 99.4(97.4) Structure and Refinement Resolution range (A) 20-1.60 15-1.66 # reflections 39,046 34,975 Rfree/Rwork (%)+ 23.0/19.0 23.3/21,2 # atoms 2,925 3304 Protein 2,925 2,826 Solvent 307 385 Mean B value overall (A2) 18.5 17.8 RMSD Bond length (A) ,0.011 0.011 RMSD Bond angle (deg) 1.35 1.49 Table 5.3. The crystal refinement statistics for 2FOX3-CexCD and 2FSX3-CexCD. The parameters denoted with (*) are defined in HKL2000, while those with (+) are defined in REFMAC. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 175 Figure 5.7. X-ray crystal structures of 2FOX3-CexCD and 2FSX3-CexCD highlighting the electron densities of the two bound inhibitors. The X-ray crystallographic structures and the 2|F0|-|FC| density (contour level at 1.0 a) of the covalently-linked inhibitors for (left) 2FOX3-CexCD and (right) 2FSX3-CexCD. For the active site sidechains and the bound inhibitors, oxygen atoms are shown coloured in red, nitrogen in blue, sulfur in yellow, and fluorine in green. The spatial orientations of the two structures are adjusted to best highlight the electron densities arising from the bound inhibitors. This figure was provided by Dr. Igor D'Angelo. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 176 and 2FSX3-CexCD, respectively). The X3 sugar rings in both 2FOX3-CexCD and 2FSX3-CexCD are solvent exposed and lack any direct or water-mediated hydrogen-bonding contacts with the enzyme. A comparison of the average X-ray crystallographic thermal factors (B-values) of the carbon atoms in each xylose moiety of 2FOX3-CexCD (XI = 15.1 A2; X2 = 15.4 A2; X3 = 30.6 A2) and 2FSX3-CexCD (XI = 17.0 A2; X2 = 15.6 A2; X3 = 29.7 A2) indeed confirm that the most distal X3 in both inhibitors are least ordered due to an absence of stabilizing interactions with the enzyme. Furthermore, within X3 for both inhibitors, the three carbons (C3, C4, and C5) furthest away from the X2 display B-values of ~ 4.0 A2 greater than those which are closest (CI and C2). ( It is also interesting to note that, when comparing the X3 residue in 2FOX3- and 2FSX3-CexCD, the sugar rings differ by a rotation of - 180° about the glycosidic X2-X3 bond. Structurally, this rotation does not result in any additional enzyme-ligand contacts. The X2-X3 orientation observed in 2FOX3-CexCD is typical of other xylooligosaccharides and xylo-thiooligosaccharides (Janis et al, 2005). Thus the orientation observed for 2FSX3-CexCD is atypical. The reason for this behaviour is uncertain. 5.4 DISCUSSION 5.4.1 The lack of a -3 glycone binding subsite in CexCD From the X-ray crystallographic structures of CexCD complexed with 2FO-DNPX3 and 2FS-DNPX3, no interactions to the X3 xylose ring and the enzyme were observed. Thus CexCD does not have a -3 glycone binding subsite. A similar conclusion may be drawn from the observation of essentially identical inactivation parameters (k; and Kj) between the xylotriosyl 2FS-DNPX3 and the xylobiosyl 2F-DNPX2 inhibitors (Table 5.2) (Tull and Withers, 1994). Prior studies suggested the presence of a -3 subsite with a small stabilization energy of ~ 1 kcal/mol (Charnock et al, 1998). This artifact may be the result of trying to thermodynamically determine small binding energies based on data obtained from the frequencies of enzymatic bond cleavage in oligosacchrides. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 177 Using disaccharide substrates with activated aryl leaving groups, the rate-determining step for Cex is deglycosylation whereas glycosylation is rate-limiting for monosaccharide derivatives (Tull and Withers, 1994). The distal sugar ring forms -2 subsite interactions to increase the rate of formation of the glycosyl-enzyme intermediate. The resulting increase in the second order rate constant, Km/Km, without significant effect on kc a t , may be the result of the increasing degree of nucleophilic pre-association by the enzymatic nucleophile and the greater extent of proton donation from the acid catalyst (Tull and Withers, 1994; Wicki et al., 2007a). Accordingly, the use of inhibitors consisting of additional sugar moieties even further away from the enzymatic cleavage site is unlikely to have additional effects on the kj. Indeed, as shown from structural and kinetic experiments presented in this chapter, the third xylose moiety in these inhibitors provides no additional enhancement to the inactivation of Cex at either the level of binding (Kj) or glycosylation (kj). 2FO-DNPX3 appears to be an ineffective inhibitor as it is hydrolyzed into inactive, non-inhibiting forms by CexCD (Figure 5.8). Since Cex remains active at low inhibitor concentrations, the formation of the 2F-xylotriosyl-CexCD complex must be slow under assay conditions. Although a Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 178 Glu233 Figure 5.8. Potential hydrolysis pathways of the xylotriose-derived inhibitor 2FO-DNPX3. All the sites of potential hydrolysis are denoted using red arrows, and the corresponding numerical designations point to the products and other subsequent reactions. Hydrolysis reactions which involve the binding of only one xylose moiety in the glycone region are unlikely to occur based on Table 5.1 (light grey). Therefore, pathway 2 (green) leads to depletion of 2FO-DNPX3 by its hydrolysis to X2 and 2F-DNPX during kinetic inactivation experiments. However, inhibition via pathway 3 (blue) does occur, leading to the crystallographically bound 2F-xylotrioside. Inhibited forms of CexCD, as determined by the formation of stable glycosyl-enzyme intermediates, are underlined. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 179 variety of hydrolysis pathways are possible, preference is given to the occupancy of both the -1 and -2, rather than just the -1, subsite ( k c a t / K m for the hydrolysis of PNPX2 is ~ 17000-fold higher than for PNPX (Table 5.1)) (Notenboom et al, 1998; Tull and Withers, 1994; Withers et al, 1990). Therefore, the most likely pathway is the cleavage of 2FO-DNPX3 into xylobiose and 2F-DNPX, with the latter also being a very inefficient inhibitor. In contrast to the results of inactivation assays, the crystal structure of 2FOX3-CexCD showed complete inhibition of CexCD with.the xylotriose inhibitor. This may be the result of the slow accumulation of the bound covalent species (24 hours), or may be attributed to using excess amounts of the inhibitor during soaking of the CexCD crystal (~ 5 mM) at lower pH (4.6). To the best of our knowledge, structures of family TO xylanases complexed with thio-oligosaccharides are unavailable. However, with the family 11 xylanases, the majority of the bound thio-oligosaccharide complexes generally adopt the low-energy 4 Q conformations (Janis et al, 2005): In comparison, Bex and the B. agaradhaerens endoglucanase Cel5A distort the naturally occurring (^ -oligosaccharides at the -1 site to the 2 , 5 B conformation (Varrot et al, 2001), thus resulting in the substrate being able to approximate the geometries of the reaction intermediates and transition states. Based on these results, we.do not expect cleavage of the thioglycoside linkage for possible geometric reasons, as well as the fact that the S.O acetal will not benefit from general acid catalysis as this would require binding to only the -1 subsite. However, pathway 2 of Figure 5.8 is also apparently excluded. The increase in the glycosidic C-S bond length and the decrease in its bond angle versus those of a C-0 bond may be sufficient to displace the orientation of the X3 ring, thereby preventing favourable binding interactions with the -2 subsite. 5.4.2 Comparison of the Cex binding site topology with those of other family 10 xylanases Structural variations in the active site binding cleft of family 10 glycosyl hydrolases can result in drastic changes to their activities and modes of action towards substrates. There are currently Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 180 365 enzymes classified as family 10 glycosyl hydrolases (www.cazy.org), and several of them have been exhaustively studied in terms of structure, substrate binding, catalysis, and stability. It is therefore impractical to attempt to compare each member in order to highlight topological variations in substrate binding. Thus, a selected few will be used for the following discussions in the context of describing similarities and differences in comparison to Cex. 5.4.2.1 The glycone (-) binding region The "substrate recognition" area for the family 10 glycoside hydrolases ranges from one (Pell et al, 2000b) to three (Andrews et al, 2000) binding sites, and can accommodate multiple types of substrates (e.g. xylan and cellulose). Specific features must therefore be present to account for these structural variations. In the -1 glycone binding pocket, a conserved tryptophan lies perpendicular to the xylose plane, effectively "behind" the C5-05 bond. In order for the enzymes to accommodate glucoside substrates, this tryptophan must rotate away from the additional C6-06 exocyclic substituent (Pell et al, 2004a). Indeed, this movement, upon cellobioside binding, is observed for both Cex (Figure 5.9) (White et al, 1996), and the Streptomyces lividans family 10 xylanase 5/Xynl0A (Ducros et al, 2000). The rotation of the tryptophan in 57XynlOA, however, is hindered by an adjacent arginine, which accounts for its decreased activity towards glucoside substrates (Ducros et al, 2000). Such movement is not required for xylose43ased substrates, thus accounting for high xylanase activities. In contrast, the xylanase from Cellvibrio japonicus (formerly Pseudomonas cellulosa), Q'XynlOA, also contain a tryptophan closely interacting with a leucine. Unlike 57XnlOA, this leucine does not sterically hinder glucoside binding, as evidenced by the lack of increased affinity for such substrates when it is removed through mutagenesis (Andrews et al, 2000). Furthermore, the Cellvibrio japonicus xylanase 10C, Q'XynlOC, contains a tryptophan that is conformationally stabilized by two leucine residues, which are in turn interacting with an additional tryptophan. This highly linked network would explain the abnormally high xylo-to-gluco activity ratio displayed by QXynlOC when compared with Q'XynlOA, 5/Xynl0A, or Cex (Pell et al, 2004a). Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 181 Figure 5.9. Position of Trp281 in 2FX2-CexCD and 2FCb-CexCD. The ribbon representation of the X-ray derived crystal structure of the inhibited (left) 2FX2-CexCD (Notenboom et ai, 1998b) and (right) 2FCb-CexCD (White et al, 1996), with their respective covalently-bound catalytic nucleophile shown in blue. Trp281 (red), which forms a part of the -1 binding subsite, rotates away from the additional hydroxymethyl group found in the cellobiosyl inhibitor. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 182 The necessity for amino acid sidechains to rotate away from bulky substrates also extends to the -2 binding subsite in the glycoside hydrolase family 10 enzymes. In S/XynlOA, Gln87 is displaced to accommodate the bulky C6 hydroxymethyl groups of glucopyranoside units (Ducros et al, 2000). This phenomenon is most likely observed also in Cex, as the equivalent glutamine sidechain become disordered in the X-ray structure of 2FCb-CexCD (White et al, 1996). Instead of a glutamine, QXynlOA has a tyrosine (Tyr87) at the same location, and this additional steric constraint is believed to contribute to the dramatically decreased rate of hydrolysis of PNPC versus PNPX2. Indeed, as shown through mutagenesis, its deletion increases both the rate of reaction and binding affinity for PNPC (Andrews et al, 2000). Likewise, QXynlOC has an equivalent tyrosine (Tyr340) which plays similar roles in catalysis and substrate binding (Pell et al, 2004a). Another well conserved active site feature found in Cex, STXynlOA, and QXynlOA, is the presence of a glutamate in the -2 subsite that is responsible for making hydrogen bonds with the C2-OH in substrates and/or inhibitors. QXynlOC lacks this moiety (replaced with a glycine) and hence is less active towards shorter xylooligosaccharides (Pell et al, 2004a). The importance of this residue was corroborated with the enzyme QXynlOA, where a glutamate-to-glycine mutation severely decreased its ability to hydrolyze the afore-mentioned xylo-oligosaccharides (Charnock et al. 1997; Charnock et al, 1998). By having a glycine in this crucial binding site, a large cavity is created in the -2 binding site allowing decorated substrates to be accommodated. Indeed, experiments have shown that xylose decorated at 02 with an arabinofuranose group is readily cleaved by QXynlOC (Pell et al, 2004a). In QXynlOA and C, the sidechain - O H groups of Tyr87 and Tyr340 provide selection against binding glucopyranosyl substrates. An alternative role for the aromatic sidechains of the tyrosine residues is the formation of stabilizing ring-ring stacking interactions as a part of the -3 binding subsites of QXynlOA, Cellvibrio mixtus XynlOA (CmXynlOA), three Fibrobacter succinogenes proteins, and five xylanases from Arabidopsis thaliana (Pell et al., 2004a; www.cazy.org). Those lacking this tyrosine, and therefore not having a -3 substrate binding subsite, Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 183 include Cex, as well as the XynlOB from C. mixtus (Pell et al, 2004b) and another family 10 xylanase from Cryptococcus albidus (Biely et al, 1981). Judging from the structures of CexCD with the xylotriosyl inhibitors, it is unlikely that this enzyme can be easily engineered to introduce a new (-3) binding subsite, as all the nearby amino acids are all pointing away from the distal xylosyl ring and would not be properly oriented to form productive ring-stacking interactions. 5.4.2.2 The aglycone (+) binding region As with the glycone (-) binding region, a large number of residues that form the aglycone, or "product release area" (Schmidt et al, 1999) are conserved amongst the family 10 glycoside hydrolase family of enzymes. The sizes of these binding regions vary with as many as four found in C/nXynlOB (Pell et al, 2004b) and Q'XynlOA (Leggio et al, 2000) in X-ray crystallographic structures of xylopentaose complexes. When substrates are bound to this area, their electron densities are often poorly defined, suggestive of conformational disorder. Thus aglycone binding may not be as specific as in the glycone region (Schmidt et al, 1999). It can be rationalized that most, but not all of these binding interactions, are mediated by the non-specific hydrophobic interactions of Phe or Tyr residues "stacking" against the pyranosyl rings of the substrates (Charnock et al, 1998; Pell et al, 2004b; Schmidt et al, 1999). The relatively indiscriminate nature of these interactions was highlighted when the Y434F substitution in Q'XynlOC and the corresponding F181Y change in Q'XynlOA produced little effect on the catalytic properties of these enzymes (Pell et al, 2004a). Charnock et al. (1998) proposed that Cex contains two (+) binding subsites, and crystallographic studies have shown that indeed, this xylanase has 4'well-conserved residues in the +1 and +2 binding areas to facilitate substrate binding (Figure 5.10). In the +1 subsite, Tyrl71 is well positioned to form a ring-stacking interaction with substrates, while Asnl72 is poised to hydrogen bond with the xylopyranoside ring in the +2 subsite. However, Tyrl71 appears to be stabilized by two additional, potentially conserved charged residues, Aspl31 and Lysl79. As these two residues are not Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 184 Figure 5.10. Bound DNP leaving group in the X-ray crystal structure of 2FOX3-CexCD. From the X-ray crystal structure of covalently-inhibited 2FOX3-CexCD, the cleaved DNP leaving group (bright green) can be observed in the +1 aglycone binding subsite of the enzyme, stacked over Tyrl71. Asnl72, which forms the +2 binding site, and two conserved residues (Aspl31, Lysl79) which most likely play important structural roles in supporting the aglycone binding sites, are also shown. Selected oxygen and nitrogen atoms are shown in red and blue, respectively. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 185 in hydrogen-bonding contact with Tyrl71, their roles may be to provide scaffolding. Their removal by mutagenesis in Q'XynlOA resulted in a decrease in the +2 site's binding capacity towards substrates with higher degrees of polymerization (Charnock et al, 1998). Unlike the P. simplicissimum xylanase and Cex, the +1 site in OnXynlOB contains multiple hydrophobic residues forming 2 hydrophobic walls with a xylopyranose ring sandwiched between Phe336 and Phe340 on one side, and Tyr200 on the other (Pell et al, 2004b). This enzyme can also utilize glutamates to hydrogen bond to substrates forming additional (+3) subsites. Furthermore, some enzymes also make use of divalent metal ions to help stabilize this binding region (Charnock et al, 1998). A special feature observed with Cex is that it is one of the most efficient at hydrolyzing aryl-glycosides amongst family 10 glycosyl hydrolases. In comparison with Q'XynlOA, it is > 30-fold more reactive towards PNPX2 (Andrews et al, 2000; Harris et al, 1994; van Tilbeurgh et al, 1982), and > 400-fold towards PNPC (Charnock et al, 1997; Charnock et al, 1998). In contrast with xylotriose, which sits at the -2 to +1 binding subsites for both enzymes, their activities are similar (Andrews et al, 2000). These observations lead to the conclusion that the Cex +1 site can better accommodate and form a tighter binding pocket for the aryl leaving groups found in many synthetic substrates and inhibitors. Consistent with this, the X-ray crystal structures of CexCD soaked with the 2FO-DNPX3 and 2FS-DNPX3 inhibitors showed cleaved DNP groups residing in the +1 binding subsite and interacting with Tyrl71 (Figure 5.10). Upon closer examination, the DNP hydroxyl group is rotated away from the anomeric centre of the covalently-bound xylotriose. This is likely the result of a change in position after hydrolysis. However, it is tempting to hypothesize that hydrolysis is facilitated by the presence of a higher-energy substrate intermediate which would have strained ring conformations to accommodate the observed atypical Cl-to-DNP bond angle. Chapter 5 - Probing for the -3 Glycone Binding Subsite in Cex 186 5.5 CONCLUSION The xylotriosyl mechanism based 2FO-DNPX3 and 2FS-DNPX3 inhibitors were synthesized and used to explore the interactions between trisaccharides and the glycone binding sites of the family 10 xylanase Cex. X-ray crystallographic structures were solved showing that the enzyme only forms stabilizing interactions with the 2 proximal xylose residues (XI and X2) in the -2 and -1 subsites. In both cases, the distal xylose ring (X3) was extended into the aqueous exterior of the active site, exhibiting elevated B-values indicative of enhanced mobility. 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Appendix 1 205 Appendix 1 NMR Chemical Shift Table for apo-CexCD1 asidue HN (40 °C)a NH (40 °C)a c* c° C Other3 A1 T2 T3 L4 9.68 122.60 178.60 57.06 39.36 K5 6.49 119.30 177.00 59.30 32.14 E6 7.60 118.90 180.20 58.46 30.01 C6, 183.70 A7 7.58 123.30 179.10 54.03 18.34 (7.58) (123.30) A8 8.25 124.20 179.80 55.17 17.77 D9 8.91 119.60 181.40 56.93 39.64 C, 178.60 G10 7.96 108.60 174.60 46.43 . -(108.40) A11 7.40 122.90 177.70 51.51 19.74 G12 7.92 110.20 174.80 46.27 - ( R13 7.93 117.50 174.90 52.43 34.58 D14 8.82 120.20 176.00 53.34 42.53 C r, 177.30 F15 10.06 125.20 175.00 56.65 41.56 (10.05) (125.10) G16 9.21 114.90 171.50 44.99 -F17 7.19 113.70 172.30 53.62 41.97 A18 5.77 119.80 174.60 49.02 15.62 L19 8.59 125.20 176.30 53.85 43.71 D20 7.07 127.10 175.50 49.97 41.77 P21 - - 177.50 63.81 31.11 N22 8.09 118.20 177.00 55.15 37.46 C\ 176.50; H 5 2, (6.97, 7.98); N 6 2, 115.90 R23 7.54 115.10 176.30 54.06 25.56 (7.56) (115.20) L24 6.85 116.40 176.20 56.49 40.55 S25 6.98 110.70 173.80 58.63 63.00 E26 7.75 126.10 176.00 55.27 28.52 C5, 184.30 A27 8.75 131.40 180.60 55.82 18.06 (8.71) (131.40) C r, 34.49; C 6, 180.5; hf2, (6.90, 7.54); Na Q28 8.89 115.50 177.40 58.59 28.36 Y29 6.46 119.40 176.60 60.91 37.30 (6.48) (119.30) K30 8.01 118.80 176.80 59.04 32.10 A31 7.92 118.20 181.30 54.72 17.71 I32 6.83 120.50 176.40 64.47 38.01 A33 7.95 123.80 179.40 54.55 18.52 D34 8.69 112.50 178.80 57.05 40.48 C\ 178.80 S35 7.46 111.80 176.50 60.13 66.05 (7.49) (118.80) E36 8.25 117.30 173.10 58.03 28.75 C5, 182.50 1 List of assigned chemical shifts for apo-CexCD at pH 6.5 and 30 °C. a Assignments of the 1 3 C y / s resonances for Asx and Glx were performed at 40 °C, with the corresponding i+l amide resonances at 40 °C denoted with (). Appendix 1 206 Residue H N N H C C" C p Other (40 °C) (40 °C) F 3 7 6 . 4 1 1 0 9 . 9 0 1 7 6 . 5 0 5 6 . 1 8 4 4 . 2 4 N 3 8 ( 6 . 4 4 ) 9 . 4 2 ( 1 0 9 . 9 0 ) 1 1 8 . 7 0 1 7 4 . 5 0 5 2 . 0 3 4 0 . 0 4 C \ 1 7 6 . 7 0 ; H 5 2 , ( 6 . 8 7 , 8 . 1 7 ) ; N 5 2 , 1 0 9 . 9 0 L 3 9 7 . 5 9 1 2 2 . 5 0 1 7 3 . 1 0 5 4 . 5 2 4 3 . 0 9 V 4 0 ( 7 . 6 1 ) 8 . 9 5 ( 1 2 2 . 5 0 ) 1 2 4 . 1 0 1 7 0 . 5 0 5 9 . 0 0 3 5 . 5 0 V 4 1 7 . 7 4 1 2 5 . 5 0 1 7 3 . 4 0 5 7 . 8 3 3 5 . 8 9 A 4 2 8 . 9 4 1 2 8 . 5 0 • 1 7 6 . 2 0 5 0 . 9 8 1 9 . 6 0 E 4 3 7 . 7 1 1 2 8 . 6 0 1 7 5 . 8 0 5 8 . 8 4 2 9 . 7 8 N 4 4 A 4 5 - - 1 7 6 . 2 0 5 4 . 8 6 1 9 . 5 2 M 4 6 9 . 0 1 1 1 3 . 5 0 1 7 4 . 8 0 5 5 . 4 6 3 2 . 4 4 K 4 7 7 . 0 4 1 1 8 . 5 0 1 7 9 . 4 0 5 7 . 0 2 3 5 . 0 7 H 5 , 7 . 9 9 ; N 5 , 3 5 . 8 9 W 4 8 1 0 . 2 2 1 2 7 . 4 0 1 7 8 . 5 0 5 9 . 9 8 2 8 . 2 0 H E 1 , 8 . 3 8 ; N E \ 1 2 5 . 4 0 D 4 9 8 . 7 3 1 1 9 . 3 0 1 7 6 . 9 0 5 6 . 9 8 3 7 . 7 0 C , 1 7 9 . 2 0 A 5 0 7 . 3 0 1 2 2 . 5 0 1 7 8 . 6 0 5 2 . 9 5 2 0 . 3 0 / T 5 1 ( 7 . 2 8 ) 7 . 8 3 ( 1 2 2 . 2 0 ) 1 0 4 . 6 0 1 7 5 . 7 0 6 1 . 8 1 7 2 . 6 9 E 5 2 8 . 8 9 1 2 4 . 1 0 1 7 2 . 9 0 5 2 . 8 1 3 1 . 2 1 P 5 3 - - 1 7 7 . 3 0 6 4 . 5 9 3 1 . 8 4 S 5 4 7 . 3 0 1 1 2 . 5 0 1 7 1 . 8 0 5 6 . 2 6 6 4 . 8 0 Q 5 5 , 7 . 3 1 1 2 3 . 7 0 1 7 7 . 1 0 5 7 . 7 6 2 6 . 0 0 C 3 2 . 2 0 ; C 6 , 1 7 9 . 1 0 ; H C 2 , 6 . 5 9 , 7 . 93 ) ; N 8 2 , 1 1 0 . 3 0 N 5 6 1 0 . 6 1 1 2 7 . 3 0 1 7 3 . 8 0 5 3 . 8 4 3 8 . 4 6 C V , 1 7 8 . 5 0 ; H 8 2 , ( 6 . 8 1 , 7 . 4 6 ) ; N 6 2 , 1 1 3 . 2 0 S 5 7 ( 1 0 . 5 9 ) 7 . 3 6 ( 1 2 7 . 1 0 ) 1 1 7 . 3 0 1 7 2 . 6 0 5 6 . 0 3 6 3 . 1 7 F 5 8 ( 7 . 3 9 ) 8 . 1 0 ( 1 1 7 . 3 0 ) 1 2 2 . 8 0 1 7 6 . 8 0 5 6 . 8 4 4 0 . 5 9 S 5 9 8 . 5 6 1 1 9 . 5 0 1 7 4 . 6 0 5 5 . 0 5 6 4 . 1 4 F 6 0 8 . 7 2 1 2 2 . 4 0 1 7 5 . 6 0 5 7 . 0 1 3 9 . 4 6 G 6 1 8 . 6 1 1 1 0 . 1 0 1 7 6 . 4 0 4 8 . 1 9 -A 6 2 8 . 7 4 1 2 5 . 3 0 1 8 0 . 3 0 5 5 . 3 8 1 7 . 7 8 G 6 3 9 . 1 0 1 0 9 . 3 0 1 7 5 . 8 0 4 7 . 4 1 -D 6 4 9 . 9 2 1 2 4 . 7 0 1 7 9 . 5 0 5 7 . 3 8 3 9 . 1 3 C , 1 7 9 . 6 0 R 6 5 7 . 9 0 1 2 2 . 2 0 1 8 0 . 0 0 5 9 . 2 1 2 9 . 5 3 V 6 6 7 . 5 1 1 2 1 . 1 0 1 7 6 . 7 0 6 6 . 3 6 3 0 . 2 1 A 6 7 8 . 4 4 1 2 2 . 0 0 1 8 0 . 4 0 5 5 . 0 9 1 7 . 6 0 S 6 8 8 . 9 6 1 1 7 . 2 0 1 7 5 . 3 0 6 1 . 6 7 6 2 . 7 9 Y 6 9 7 . 6 3 1 2 3 . 3 0 1 7 8 . 4 0 6 1 . 9 0 3 7 . 5 3 A 7 0 7 . 9 4 1 2 2 . 8 0 1 7 8 . 4 0 5 5 . 2 2 1 7 . 2 8 A 7 1 7 . 8 7 1 1 9 . 6 0 1 8 1 . 2 0 5 4 . 4 9 1 7 . 8 8 D 7 2 8 . 6 4 1 1 9 . 2 0 1 7 8 . 3 0 5 6 . 6 2 4 0 . 6 8 C \ 1 7 9 . 1 0 T 7 3 7 . 2 9 1 0 5 . 4 0 1 7 5 . 6 0 6 0 . 8 9 6 9 . 7 1 G 7 4 ( 7 . 3 2 ) 7 . 5 8 ( 1 0 5 . 4 0 ) 1 1 3 . 9 0 1 7 5 . 1 0 4 6 . 8 6 _ K 7 5 8 . 4 1 1 2 0 . 6 0 1 7 6 . 0 0 5 5 . 0 3 3 6 . 0 2 E 7 6 7 . 7 3 1 2 1 . 8 0 1 7 4 . 3 0 5 6 . 0 5 3 0 . 7 9 C 6 , 1 8 3 . 2 0 L 7 7 8 . 7 3 1 2 4 . 9 0 1 7 4 . 0 0 5 3 . 0 6 4 3 . 0 9 Y 7 8 ( 8 . 7 3 ) 8 . 9 1 ( 1 2 4 . 9 0 ) 1 3 1 . 9 0 1 7 5 . 1 0 5 6 . 1 8 4 1 . 7 9 G 7 9 8 . 6 3 1 1 7 . 6 0 1 7 0 . 2 0 4 5 . 0 5 - . H 8 0 8 . 0 7 1 2 9 . 5 0 1 7 2 . 9 0 5 1 . 4 2 3 2 . 1 4 H 6 2 , 7 . 1 2 ; N H B 1 , 7 . 4 2 ; C E 1 , 1 3 7 . 9 0 ; N S 1 , 2 0 2 . 5 0 ; N ' 2 , 1 8 2 . 4 0 T 8 1 6 . 4 5 1 2 0 . 1 0 1 7 1 . 9 0 6 0 . 4 5 7 0 . 8 7 Appendix 1 207 Residue H N N H C C a C p Other (40 °C) (40 °C) L82 7.78 128.80 . 173.80 58.61 40.70 V83 7.59 116.20 172.80 63.59 32.36 W84 6.64 124.30 175.10 53.58 - H E\ 10.87; N s 1, 102.20 H85 H 8 2 , 6.91; C 5 2 , 118.40; H 6 \ 8.52; C b 1 , 139.20; N 5 1 247.60; Ha, 11.83; NP 2 , 182.40 S86 Q87 L88 P89 - - 176.30 62.10 32.84 D90 8.58 122.90 178.30 57.52 39.80 C\ 179.40 W91 8.21 115.80 178.00 58.61 27.26 H e 1 , 9.54; N B\ 128.30 A92 (8.17) 5.87 (115.80) 124.80 178.10 53.81 15.77 K93 6.71 115.30 176.80 58.71 31.63 N94 7.08 113.70 175.30 52.97 39.01 C, 176.80; H 6 2, (6.83, 7.34); N 5 2, 113.50 L95 7.00 120.40 175.50 53.94 43.54 N96 (7.00) 8.58 (120.30) 118.00 175.70 51.81 42.67 > C, 176.70; H 5 2, (6.86, 7.52); N 6 2, 115.70 G97 8.86 ' 108.80 175.10 46.86 -S98 (8.85) (108.80) 176.80 61.04 62.59 A99 8.29 125.90 180.80 54.76 18.21 F100 7.51 121.80 176.80 60.44 37.55 E101 8.43 121.60 178.40 60.50 29.75 C6, 182.80 S102 8.76 113.90 177.20 61.45 62.70 A103 (8.73) 7.88 (113.80) 124.90 180.70 54.78 18.07 M104 8.30 120.90 177.60 59.47 32.00 V105 8.73 120.80 179.20 67.60 31.32 N106 8.95 121.60 175.60 57.03 39.17 C\ 176.10; H 5 2, (7,35, 8.51); N 5 2, 116.70 H107 8.34 (8.36) 118.60 (118.50) 175.70 60.97 28.49 H 5 2, 6.28; G 6 2, 118.60; H B\ 8.27; C e 1, 135.80; H81, 16.74; N s 1, 184.80; HP 2 , 13.52; N c 2, 170.70 V108 8.i3 116.40 176.10 66.22 31.50 T109 8.09 112.70 175.30 66.24 68.98 K110 8.22 118.70 180.80 58.21 30.91 V111 7.73 120.00 177.10 66.81 -A112 7.75 120.60 180.90 54.98 16.34 D113 8.59 117.00 178.30 57.25 42.57 C , 179.10 H114 7.42 (7.44) 116.40 (116.40) 176.30 59.29 27.95 H 6 2 , 3.79; C 8 2 , 118.80; N H b 1 , 8.28; C E 1 , 135.70; N 5 1, 181.7; N b 2 , 177.4 F115 7.14 112.40 173.20 58.27 39.86 E116 7.21 126.20 177.50 58.45 27.99 C5, 182.40 G117 , 9.70 118.60 174.20 45.10 -K118 (9.67) 8.24 (118.50) 119.30 176.40 56.82 . 33.07 V119 7.34 117.90 174.70 61.51 31.98 A120 8.29 128.90 178.70 53.19 19.78 S121 6.57 111.30 171.00 55.92 66.95 W122 8.92 121.70 175.80 55.56 34.93 H e 1 , 12.14; N b 1 , 101.50 D123 8.77 120.20 176.30 53.52 40.36 V124 9.49 132.50 174.30 65.41 30.46 V125 (9.49) 6.46 (132.30) . 112.50 172.20 60.34 35.25 N126 8.29 127.00 173.90 50.27 42.40 C\ 174.30 Appendix 1 208 esidue H N (40 °C) N H (40 °C) c C" C E127 8.80 121.00. 174.40 56.88 26.57 (8.80) (121.00) A128 8.90 118.10 179.50 . 52.87 19.79 (8.89) (118.20) F129 7.96 117.50 173.70 59.08 40.35 A130 8.63 124.00 178.50 49.87 20.32 131 G132 - - • 174.70 44.71 -G133 7.93 108.90 . 173.50 43.56 -G134 8.28 106.70 173.70 43.96 - . R135 8.67 116.50 177.10 53.50 30.37 R136 8.08 122.90 175.60 57.12 29.35 Q137 8.63 124.20 177.10 56.11 28.57 D138 8.18 118.60 174.70 52.28 38.29 (8.23) (118.40) S139 6.49 111.90 175.30 54.14 63.64 (6.49) (111.80) A140 8.95 132.30 178.20 54.53 17.76 F141 6.96 113.50 176.10 61.16 36.71 Q142 6.81 119.80 179.10 57.27 29.33 Q143 8.33 116.60 178.50 58.39 28.36 (8.33) (116.50) K144 7.80 114.80 178.40 58.42 34.11 (7.82) (114.70) L145 7.83 115.00 178.10 53.83 42.57 G146 7.58 109.90 173.60 44.52 -N147 8.72 115.50 180.50 54.72 38.12 G148 - - 174.80 46.55 -Y149 7.13 117.50 175.40 60.03 38.65 1150 5.87 120.20 177.30 65.50 36.26 E151 8.72 122.80 177.70 59.83 27.93 T152 7.81 116.40 176.70 66.00 67.78 (7.82) (116.40) A153 8.38 124.40 178.70 55.97 17.58 F154 8.34 117.10 178.30 64.00 38.48 R155 8.49 117.00 179.60 60.42 28.72 A156 8.07 122.30 179.70 54.11 17.41 A157 8.18 123.30 177.50 55.08 17.95 R158 7.81 117.10 177.20 55.92 27.44 A159 7.32 119.20 178.30 53.58 17.56 A160 7.11 119.30 176.80 52.75 21.05 D161 7.76 114.70 173.70 50.57 41.12 P162 - - 177.40 63.68 32.59 T163 8.24 110.50 175.30 62.25 70.05 A164 7.58 126.90 178.00 52.07 18.26 K165 8.47 122.50 176.40 54.94 31.05 L166 10.41 125.20 175.80 54.13 40.55 C167 10.02 123.20 175.50 52.01 39.43 1168 8.04 121.90 171.50 . 58.55' 41.82 N169 8.07 126.60 175.50 51.74 43.16 D170 8.32 126.80 171.70 53.74 46.27 (8.32) (126.70) C s, 180.30; H s 2, (6.83, 7.14); N 8 2, 113.50 C5, 180.10 C\ 33.22; C 6, 180.00; H 6 2, (6.64, 7.32); N 6 2, 111.00 C5, 182.40 C\ 174.50 Cy, 180.60 Appendix 1 209 tesidue H N (40 °C) (40 °C) c C" C 5 Other Y 1 7 1 1 0 . 1 3 1 1 7 . 2 0 1 7 6 . 5 0 5 4 . 7 3 4 0 . 2 6 N 1 7 2 ( 1 0 . 1 5 ) 9 . 2 8 ( 1 1 7 . 2 0 ) 1 2 1 . 8 0 1 7 3 . 9 0 5 4 . 1 2 3 6 . 1 3 C T , 1 7 8 . 3 0 ; H 6 2 , ( 7 . 1 6 , 7 . 8 2 ) ; N 6 2 , 1 1 2 . 1 0 V 1 7 3 6 . 61 1 0 3 . 9 0 1 7 2 . 9 0 5 8 . 9 7 3 1 . 0 1 E 1 7 4 ( 6 . 6 4 ) 7 . 9 9 ( 1 0 3 . 9 0 ) 1 2 0 . 8 0 1 7 6 . 9 0 6 1 . 7 4 2 7 . 2 4 H R , ( 2 . 3 9 ) ; C Y , 3 6 . 4 8 ; C 5 , 1 8 3 . 8 0 G 1 7 5 7 . 4 2 1 0 3 . 2 0 1 7 3 . 1 0 4 2 . 5 1 -1176 ( 7 . 4 2 ) 8 . 1 3 ( 1 0 3 . 1 0 ) 1 1 9 . 9 0 1 7 4 . 5 0 6 3 . 0 9 3 4 . 7 8 N 1 7 7 7 . 8 8 1 2 7 . 3 0 1 7 3 . 7 0 5 1 . 5 8 3 8 . 5 9 A 1 7 8 - - 1 7 9 . 6 0 5 5 . 3 4 1 8 . 1 6 K 1 7 9 8 . 1 9 1 2 1 . 9 0 1 7 8 . 5 0 6 0 . 5 4 3 3 . 2 8 S 1 8 0 9 . 4 9 1 1 6 . 2 0 1 7 7 . 4 0 6 0 . 7 7 6 3 . 0 5 N 1 8 1 9 . 0 0 1 2 3 . 7 1 0 ' 1 7 7 . 9 0 5 5 . 5 2 3 6 . 2 6 C, 1 7 5 . 8 0 ; H 8 2 , ( 6 . 3 1 , 7 . 2 8 ) ; N 6 2 , 9 . 0 0 S 1 8 2 7 . 8 3 1 1 9 . 8 0 1 7 5 . 9 0 6 2 . 6 7 6 3 . 4 0 L 1 8 3 ( 7 . 8 3 ) 7 . 3 7 ( 1 1 9 . 8 0 ) 1 2 6 . 4 0 1 7 6 . 6 0 5 7 . 5 4 4 1 . 1 9 Y 1 8 4 8 . 9 2 1 2 2 . 3 0 1 7 6 . 1 0 6 1 . 7 8 3 7 . 4 6 D 1 8 5 7 . 9 6 1 1 8 . 7 0 1 7 9 . 2 0 5 7 . 0 0 4 0 . 1 2 C R , 1 7 9 . 0 0 L 1 8 6 7 . 3 1 1 2 2 . 7 0 1 7 7 . 5 0 5 8 . 2 7 4 0 . 7 3 V 1 8 7 ( 7 . 3 3 ) 8 . 1 8 ( 1 2 2 . 6 0 ) . 1 2 1 . 2 0 1 7 7 . 2 0 6 6 . 6 7 3 0 . 2 4 K 1 8 8 8 . 4 5 1 1 9 . 7 0 ' 1 7 8 . 9 0 5 9 . 9 1 3 1 : 9 9 D 1 8 9 , 7 . 6 3 1 2 0 . 3 0 1 7 7 . 0 0 5 7 . 0 0 4 2 . 2 7 C Y , 1 8 0 . 2 0 F 1 9 0 9 . 1 9 1 2 1 . 0 0 1 7 9 . 4 0 5 9 . 5 4 3 6 . 8 8 K 1 9 1 ( 9 . 1 8 ) 8 . 6 4 ( 1 2 0 . 9 0 ) 1 1 6 . 6 0 1 8 0 . 2 0 5 6 . 0 7 2 8 . 5 0 A 1 9 2 8 . 0 7 1 2 3 . 8 0 1 8 0 . 2 0 5 4 . 7 3 1 7 . 9 3 R 1 9 3 7 . 7 9 1 1 4 . 2 0 1 7 6 . 7 0 5 6 . 4 7 3 0 . 9 9 G 1 9 4 7 . 6 3 1 0 9 . 4 0 1 7 5 . 3 0 4 5 . 9 6 V 1 9 5 8 . 3 3 1 2 5 . 5 0 1 7 5 . 1 0 6 0 . 3 2 3 1 . 8 5 - P 1 9 6 - - 1 7 2 . 3 0 6 3 . 6 2 3 0 . 8 0 L 1 9 7 7 . 2 6 1 2 1 . 8 0 1 7 5 . 4 0 5 6 . 9 9 4 2 . 0 6 D 1 9 8 9 . 8 1 1 2 3 . 7 0 1 7 5 . 9 0 5 4 . 7 3 4 5 . 0 1 C R , 1 7 8 . 6 0 C 1 9 9 8 . 1 2 1 1 6 . 7 0 1 7 2 . 7 0 5 3 . 4 0 4 9 . 0 5 V 2 0 0 ( 8 . 1 5 ) 8 . 5 1 ( 1 1 6 . 5 0 ) 1 2 0 . 7 0 1 7 3 . 4 0 6 0 . 6 1 3 4 . 4 0 G 2 0 1 9 . 5 6 1 1 5 . 8 0 1 7 0 . 7 0 4 4 . 4 3 -F 2 0 2 8 . 4 6 1 2 2 . 4 0 1 7 8 . 0 0 5 5 . 0 7 3 8 . 7 4 Q 2 0 3 9 . 0 0 1 2 5 . 2 0 1 7 5 . 8 0 5 8 . 8 1 2 8 . 8 4 C 5 , 1 7 8 . 1 0 S 2 0 4 . 8 . 4 6 1 0 1 . 9 0 1 7 4 . 3 0 5 8 . 4 5 6 1 . 3 9 H 2 0 5 ( 8 . 4 9 ) 8 . 1 3 ( 1 0 1 . 9 0 ) 1 1 8 . 6 0 1 7 5 . 3 0 5 4 . 7 5 2 7 . 0 1 H 5 2 , 7 . 5 9 ; C 5 2 , 1 2 0 . 0 0 ; N H E 1 , 7 . 7 0 ; C E 1 , 1 3 8 . 2 0 ; N 6 1 , 1 8 2 . 8 0 ; N* 2, 1 7 1 . 2 L 2 0 6 8 . 5 8 1 2 5 . 4 0 1 7 5 . 2 0 5 2 . 8 1 4 4 . 1 2 I 2 0 7 8 . 3 8 1 2 3 . 8 0 1 7 4 . 7 0 6 0 . 1 6 4 0 . 4 3 V 2 0 8 8 . 2 4 1 2 5 . 4 0 1 7 7 . 3 0 6 2 . 4 4 3 1 . 5 4 G 2 0 9 1 1 . 8 7 1 2 0 . 0 0 1 7 6 . 4 0 4 4 . 5 8 -Q 2 1 0 8 . 3 7 1 2 0 . 8 0 1 7 7 . 1 0 5 3 . 6 0 2 7 . 9 5 C Y , 3 2 . 9 8 ; C S , 1 8 0 . 6 0 ; H E 2 , ( 6 . 6 8 , 7 . 33 ) ; Na, 1 1 2 . 3 0 V 2 1 1 8 . 8 6 1 2 6 . 9 0 1 7 5 . 6 0 6 1 . 7 7 3 1 . 4 8 P 2 1 2 ( 8 . 8 0 ) ( 1 2 6 . 7 0 ) 1 7 8 . 8 0 6 3 . 4 4 3 1 . 4 4 G 2 1 3 8 . 9 8 1 1 3 . 7 0 1 7 4 . 3 0 4 6 . 2 5 -D 2 1 4 8 . 8 9 1 1 8 . 7 0 1 7 5 . 7 0 5 1 . 7 3 3 8 . 0 9 C \ 1 8 0 . 5 0 Appendix 1 210 esidue H N (40 °C) N H (40 °C) c c° F 2 1 5 6 . 9 0 1 2 1 . 6 0 1 7 8 . 6 0 5 7 . 9 5 3 8 . 9 6 R 2 1 6 ( 6 . 9 5 ) 8 . 9 5 ( 1 2 1 . 5 0 ) 1 1 8 . 9 0 1 7 7 . 7 0 6 0 . 3 5 2 8 . 0 8 Q 2 1 7 8 . 4 3 1 1 8 . 9 0 1 7 9 . 1 0 5 9 . 2 0 2 7 . 5 4 N 2 1 8 8 . 5 5 1 2 0 . 3 0 1 7 6 . 5 0 5 5 . 7 1 3 9 . 0 0 L 2 1 9 ( 8 . 5 6 ) 8 . 6 0 ( 1 2 0 . 2 0 ) 1 1 9 . 0 0 1 7 9 . 6 0 5 8 . 1 9 4 2 . 6 5 Q 2 2 0 ( 8 . 5 8 ) 8 . 6 9 ( 1 1 8 . 9 0 ) 1 1 7 . 1 0 1 7 7 . 6 0 5 7 . 4 6 2 8 . 5 0 R 2 2 1 8 . 0 6 1 1 7 . 5 0 1 7 9 . 5 0 5 8 . 6 4 2 6 . 8 9 F 2 2 2 ( 8 . 0 8 ) 7 . 6 0 ( 1 1 7 . 5 0 ) 1 2 1 . 8 0 1 7 7 . 1 0 6 3 . 0 9 3 9 . 4 6 A 2 2 3 8 . 2 8 1 2 3 . 9 0 1 8 2 . 2 0 5 4 . 1 6 1 8 . 3 4 D 2 2 4 8 . 5 7 1 1 8 . 7 0 1 7 7 . 9 0 5 6 . 3 1 4 0 . 2 8 L 2 2 5 7 . 8 7 1 2 0 . 2 0 1 7 7 . 4 0 5 5 . 2 0 4 1 . 7 8 G 2 2 6 ( 7 . 8 8 ) 8 . 4 0 ( 1 2 0 . 1 0 ) 1 0 6 . 6 0 1 7 4 . 2 0 4 5 . 2 4 V 2 2 7 6 . 8 4 1 1 0 . 8 0 1 7 3 . 6 0 5 7 . 9 6 3 3 . 9 6 D 2 2 8 7 . 6 2 1 1 7 . 6 0 1 7 6 . 2 0 5 3 . 5 8 4 2 . 3 0 V 2 2 9 8 . 0 8 1 1 0 . 1 0 1 7 4 . 9 0 5 7 . 8 0 3 5 . 0 8 R 2 3 0 ( 8 . 1 3 ) 8 . 6 3 ( 1 1 0 . 2 0 ) 1 2 1 . 4 0 1 7 3 . 5 0 5 5 . 8 9 3 4 . 5 6 1231 7 . 9 7 1 2 8 . 7 0 1 7 7 . 0 0 6 0 . 3 0 3 7 . 6 7 T 2 3 2 8 . 0 0 1 1 8 . 0 0 1 7 4 . 8 0 6 2 . 9 8 6 9 . 0 2 E 2 3 3 7 . 4 7 1 2 3 . 8 0 1 7 3 . 3 0 5 2 . 5 4 3 1 . 4 5 L 2 3 4 7 . 9 9 1 1 5 . 7 0 1 7 8 . 0 0 5 5 . 8 5 4 1 . 7 1 D 2 3 5 ( 7 . 9 9 ) ( 1 1 5 . 7 0 ) I 2 3 6 - - 1 7 5 . 0 0 5 9 . 9 9 3 7 . 6 6 R 2 3 7 8 . 4 5 1 2 3 . 2 0 1 7 3 . 7 0 5 4 . 8 3 3 2 . 4 5 M 2 3 8 8 . 4 0 1 1 7 . 8 0 1 7 4 . 7 0 5 2 . 4 2 3 7 . 0 1 R 2 3 9 8 . 5 6 1 2 1 . 3 0 1 7 7 . 0 0 5 5 . 7 6 2 8 . 7 1 T 2 4 0 7 . 9 9 1 1 7 . 7 0 1 7 3 . 1 0 5 9 . 4 1 6 7 . 1 7 P 2 4 1 S 2 4 2 D 2 4 3 A 2 4 4 - - 1 8 0 . 7 0 5 5 . 6 8 1 8 . 2 5 T 2 4 5 8 . 2 0 1 1 7 . 9 0 1 7 7 . 3 0 6 6 . 1 2 6 8 . 2 2 K 2 4 6 8 . 9 8 1 2 5 . 6 0 1 7 9 . 9 0 6 0 . 1 1 3 4 . 7 9 L 2 4 7 8 . 4 3 1 1 7 . 3 0 1 8 0 . 3 0 5 7 . 6 5 4 1 . 6 5 A 2 4 8 7 . 9 0 1 2 5 . 0 0 1 8 1 . 0 0 5 4 . 8 1 1 7 . 6 0 T 2 4 9 8 . 4 4 1 2 0 . 9 0 1 7 5 . 0 0 6 6 . 3 6 6 8 . 2 0 Q 2 5 0 8 . 6 1 1 2 0 . 3 0 1 7 7 . 5 0 5 8 . 8 7 3 0 . 3 3 A 2 5 1 7 . 9 1 1 2 2 . 2 0 1 7 7 . 4 0 5 5 . 7 9 1 7 . 4 2 A 2 5 2 7 . 5 1 1 2 0 . 7 0 1 8 0 . 6 0 5 4 . 6 4 1 7 . 4 5 D 2 5 3 8 . 4 8 1 2 3 . 2 0 1 7 8 . 0 0 5 7 . 6 6 4 1 . 2 7 Y 2 5 4 K 2 5 5 K 2 5 6 - -' 1 8 0 . 0 0 5 9 . 4 3 3 2 . 6 1 V 2 5 7 7 . 9 2 1 2 0 . 6 0 1 7 6 . 7 0 6 6 . 5 8 3 1 . 1 1 V 2 5 8 7 . 8 4 1 1 8 . 3 0 1 7 8 . 6 0 6 6 . 7 8 2 9 . 8 7 Q 2 5 9 8 . 8 0 1 1 8 . 9 0 1 7 7 . 5 0 5 9 . 3 6 2 8 . 3 4 C , 3 3 . 7 6 ; C 5 , 1 8 0 . 1 0 ; H S 2 , ( 6 . 8 3 , 7 . 47 ) ; N T 1 1 2 . 3 0 C \ 1 7 3 . 8 0 W 3 3 . 2 4 ; C 8 , 1 8 0 . 3 0 ; H 6 2 , ( 7 . 0 0 , 7 . 99 ) ; N B 2 , 1 1 8 . 9 0 C 1 7 9 . 2 0 C \ 1 7 8 . 8 0 C 5 , 1 8 2 . 6 0 C 6 , 1 7 7 . 6 0 C \ 3 4 . 8 6 ; C 5 , 1 8 0 . 2 0 ; Ha, ( 6 . 76 ) ; N , 1 1 2 . 5 0 Appendix 1 211 Residue H N (40 °C) N H (40 °C) c C" Other A 2 6 0 7 . 5 4 1 2 1 . 7 0 1 7 7 . 9 0 5 5 . 2 4 1 6 . 2 3 C 2 6 1 ( 7 . 5 7 ) 6 . 9 0 ( 1 2 1 . 7 0 ) 1 1 1 . 5 0 1 7 6 . 9 0 5 7 . 5 9 4 1 . 3 2 M 2 6 2 8 . 3 8 1 1 8 . 5 0 1 7 8 . 6 0 5 5 . 7 5 3 0 . 0 5 Q 2 6 3 7 . 5 5 1 1 6 . 9 0 1 7 4 . 9 0 5 6 . 0 9 2 8 . 9 3 C, 3 4 . 2 1 ; C S , 1 8 0 . 3 0 ; Ha, ( 6 . 6 1 , 7 . 1 6 ) ; N 0 2 , 1 1 0 . 6 0 V 2 6 4 7 . 6 3 1 2 4 . 2 0 1 7 7 . 0 0 6 1 . 1 5 3 2 . 7 5 T 2 6 5 ( 7 . 6 4 ) 9 . 0 9 ( 1 2 4 . 0 0 ) 1 2 4 . 0 0 1 7 6 . 4 0 6 6 . 0 3 6 8 . 3 7 R 2 6 6 7 . 2 8 114.40 1 7 6 . 6 0 5 5 . 9 7 2 9 . 2 3 C 2 6 7 8 . 1 7 1 2 2 . 5 0 1 7 4 . 6 0 5 5 . 5 0 4 4 . 7 6 Q 2 6 8 9 . 2 7 1 2 8 . 6 0 1 7 6 . 4 0 5 6 . 1 1 3 0 . 1 3 C, 3 3 . 8 6 ; C 5 , 1 7 8 . 4 0 ; Ha, ( 6 . 8 7 , 6 . 9 1 ) ; NP2, 1 1 4 . 7 0 G 2 6 9 7 . 3 3 1 0 1 . 5 0 1 6 9 . 7 0 4 5 . 5 0 -V 2 7 0 ( 7 . 3 4 ) 8 . 3 0 ( 1 0 1 . 5 0 ) 1 1 8 . 9 0 1 7 3 . 1 0 6 0 . 3 3 3 6 . 7 6 T 2 7 1 8 . 6 7 1 2 8 . 4 0 1 7 2 . 6 0 5 9 . 6 8 7 0 . 1 4 V 2 7 2 8 . 3 7 1 1 9 . 7 0 1 7 8 . 2 0 5 8 . 6 3 3 3 . 5 1 W 2 7 3 8 . 9 8 1 3 0 . 2 0 1 7 5 . 4 0 5 5 . 4 9 2 7 . 5 1 H E 1 , 9 . 6 6 ; N E 1 , 1 2 8 . 0 0 G 2 7 4 8 . 2 0 1 1 1 . 8 0 1 7 1 . 6 0 4 2 . 8 2 -I 2 7 5 8 . 7 7 1 1 5 . 9 0 1 7 4 . 7 0 6 3 . 3 9 4 0 . 6 4 T 2 7 6 7 . 1 5 1 0 9 . 8 0 1 7 4 . 3 0 5 6 . 0 7 6 9 . 5 4 D 2 7 7 8 . 4 1 1 3 1 . 1 0 1 7 7 . 5 0 5 6 . 9 8 4 1 . 8 8 C \ 1 7 5 . 9 0 K 2 7 8 7 . 8 0 1 1 9 . 2 0 1 7 6 . 2 0 5 8 . 9 8 3 3 . 3 9 Y 2 7 9 ( 7 . 7 9 ) 7 . 2 2 ( 1 1 9 . 0 0 ) 1 1 5 . 8 0 1 7 4 . 8 0 5 4 . 4 0 3 8 . 6 4 S 2 8 0 7 . 1 7 1 1 4 . 1 0 1 7 5 . 6 0 5 7 . 0 2 6 4 . 2 8 W 2 8 1 8 . 7 8 1 2 9 . 0 0 1 7 6 . 6 0 5 8 . 4 0 2 8 . 1 1 H C 1 , 11.93; N B 1 , 1 0 1 . 8 0 V 2 8 2 6 . 7 5 1 1 9 . 8 0 1 7 4 . 1 0 6 7 . 5 6 -P 2 8 3 - - 1 7 7 . 8 0 6 4 . 9 8 3 0 . 6 2 D 2 8 4 7 . 2 3 1 1 4 . 8 0 1 7 6 . 8 0 5 5 . 7 8 4 0 . 8 2 C \ 1 7 9 . 8 0 V 2 8 5 7 . 1 4 1 1 9 . 2 0 1 7 4 . 7 0 6 3 . 2 2 3 3 . 3 0 F 2 8 6 ( 7 . 1 7 ) 8 . 4 0 ( 1 1 9 . 2 0 ) 1 2 2 . 0 0 1 7 1 . 4 0 5 4 . 1 8 3 8 . 2 6 P 2 8 7 - - 1 7 9 . 3 0 6 3 . 4 6 3 1 . 0 6 G 2 8 8 8 . 8 3 1 1 2 . 5 0 1 7 3 . 7 0 4 4 . 8 0 -E 2 8 9 8 . 3 1 1 2 0 . 1 0 1 7 6 . 4 0 5 3 . 9 2 3 2 . 9 8 C 5 , 1 8 2 . 7 0 G 2 9 0 8 . 7 7 1 0 4 . 8 0 1 7 0 . 9 0 4 6 . 3 6 -A 2 9 1 ( 8 . 7 6 ) 8 . 2 3 ( 1 0 4 . 6 0 ) 1 2 4 . 3 0 1 7 8 . 0 0 5 2 . 0 9 1 7 . 7 9 A 2 9 2 8 . 8 9 . 1 2 0 . 9 0 1 7 8 . 9 0 5 3 . 8 1 2 2 . 5 0 L 2 9 3 9 . 4 6 1 2 3 . 4 0 1 7 7 . 0 0 5 4 . 3 4 4 1 . 4 1 V 2 9 4 8 . 0 6 1 1 9 . 4 0 1 7 3 . 3 0 6 5 . 4 2 3 0 . 2 8 W 2 9 5 7 . 3 6 1 1 6 . 6 0 1 7 6 . 2 0 5 8 . 0 4 3 2 . 5 0 H C 1 , 1 0 . 0 9 ; N E \ 1 3 0 . 5 0 D 2 9 6 8 . 7 3 1 2 0 . 1 0 1 7 9 . 6 0 5 2 . 0 5 4 0 . 2 4 A 2 9 7 - - 1 7 5 . 8 0 5 4 . 2 0 1 7 . 9 1 S 2 9 8 8 . 4 4 1 1 4 . 5 0 1 7 4 . 0 0 5 7 . 3 4 6 3 . 9 0 Y 2 9 9 7 . 9 3 1 1 3 . 7 0 1 7 3 . 4 0 6 1 . 9 2 3 3 . 4 3 A 3 0 0 8 . 4 7 1 2 3 . 6 0 1 7 8 . 4 0 5 0 . 8 6 1 8 . 8 4 K 3 0 1 8 . 4 3 1 2 2 . 2 0 1 7 7 . 6 0 5 7 . 2 6 3 2 . 7 5 K 3 0 2 7 . 7 5 1 2 5 . 8 0 1 7 6 . 3 0 5 5 . 0 5 2 9 . 5 3 P 3 0 3 - - 1 7 7 . 0 0 6 5 . 3 7 3 0 . 5 6 A 3 0 4 7 . 5 7 1 1 7 . 3 0 1 7 6 . 2 0 5 4 . 4 1 1 9 . 3 9 Y 3 0 5 7 . 8 6 1 1 8 . 3 0 1 7 6 . 0 0 6 2 . 4 6 3 7 . 6 2 Appendix 1 212 Residue (40 °C) N (40 °C) C p Other A306 7.63 120.30 179.60 53.91 17.91 A307 6.96 120.60 179.20 54.50 20.25 V308 7.80 119.60 177.00 66.72 30.45 M309 7.46 118.70 178.60 59.11 31.85 E310 8.05 117.90 180.50 58.18 29.44 C5, 183.30 A311 8.10 124.10 179.00 54.33 19.75 (8.11) (124.10) F312 7.56 115.10 176.70 59.32 39.57 G313 7.83 107.00 173.90 45.09 -A314 7.47 123.60 176.60 51.12 19.82 S315 8.11 116.70 172.80 55.89 63.69 Appendix 2 213 Append ix 2 N M R C h e m i c a l S h i f t T a b l e f o r 2 F C b - C e x C D 2 ssidue HN NH C" (40 °C) (40 °C) A1 T2 - - 62.64 69.82 T3 7.30 110.80 58.78 74.19 L4 9.69 122.50 57.03 39.31 K5 6.49 119.30 . 59.31 32.15 E6 7.62 118.80 58.45 29.97 A7 7.60 123.20 54.02 18.36 (7.61) (123.20) A8 8.27 124.20 55.21 17.70 D9 8.93 119.60 56.92 39.56 G10 7.98 108.70 46.43 -(8.00) (108.60) A11 7.41 122.90 51.52 19.71 G12 7.93 110.10 46.27 -R13 7.94 117.40 52.41 34.64 D14 8.89 120.30 53.29 42.53 F15 10.11 125.40 56.68 . 41.53 (10.11) (125.50) G16 9.21 114.80 44.98 -F17 7.14 113.50 53.52 41.92 A18 5.61 119.70 48.98 14.94 L19 8.56 125.20 53.90 43.47 D20 7.13. 127.40 49.95 41.76 P21 - - 63.83 30.96 N22 8.10 118.10 55.18 37.46 R23 7.56 115.10 54.05 25.54 (7.57) (115.10) L24 6.86 116.50 56.48 40.46 S25 6.99 110.70 58.65 63.01 E26 7.76 126.00 55.27 28.45 A27 8.75 131.30 55.79 18.03 (8.72) (131.30) Q28 8.90 115.40 58.58 28.38 Y29 6.46 119.40 60.95 37.25 (6.48) (119.30) K30 8.01 118.70 59.09 31.94 A31 7.93 118.20 54.73 17.70 I32 6.85 120.50 64.46 37.99 A33 7.97 123.80 54.57 18.51 D34 8.71 112.50 57.12 40.46 S35 7.47 111.80 60.17 66.03 (7.51) (111.90) E36 8.27 117.20 57.97 28.83 F37 6.42 109.90 56.19 44.11 (6.45) (109.90) C8, 183.70 C, 178.70 C\ 177.30 C 176.50; H 8 2, (6.96, 7.99);N62, 116.00 C8, 184.40 CT, 34.43; C8, 180.50; HP2, (6.69; 7.54); N 8 2, 113.60 C, 178.80 C8, 182.50 2 List of assigned chemical shifts for 2FCb-CexCD at pH 6.5 and 30 °C. a Assignments of the 1 3 G T / 5 resonances for Asx and Glx were performed at.40 °C, with the corresponding i+l amide resonances denoted with (). Appendix 2 214 esidue H N N H c a c p (40 °C) (40 °C) N 3 8 9 . 3 6 1 1 8 . 7 0 5 2 . 0 0 3 9 . 9 8 L 3 9 7 . 6 0 1 2 2 . 4 0 5 4 . 5 0 4 3 . 0 9 ( 7 . 6 3 ) ( 1 2 2 . 6 0 ) V 4 0 8 . 9 5 1 2 4 . 1 0 5 9 . 0 0 3 5 . 4 7 V 4 1 7 . 7 2 1 2 5 . 6 0 5 7 . 6 9 3 5 . 8 5 A 4 2 8 . 9 6 1 2 8 . 6 0 5 1 . 1 9 1 9 . 4 0 E 4 3 7 . 8 5 1 2 8 . 8 0 5 8 . 6 8 2 9 . 5 5 N 4 4 8 . 5 8 1 0 8 . 9 0 5 4 . 6 1 4 3 . 9 6 ( 8 . 5 9 ) ( 1 0 8 . 9 0 ) A 4 5 7 . 2 1 1 1 8 . 6 0 5 4 . 7 8 1 9 . 5 8 ( 7 . 2 2 ) ( 1 1 8 . 6 0 ) M 4 6 9 . 0 1 1 1 3 . 6 0 5 5 . 4 3 3 2 . 1 4 K 4 7 6 . 9 7 1 1 8 . 5 0 5 7 . 3 4 3 4 . 8 5 W 4 8 1 0 . 1 8 1 2 7 . 1 0 5 9 . 9 3 2 8 . 2 0 D 4 9 8 . 8 6 1 1 9 . 1 0 5 6 . 9 7 3 8 . 1 3 A 5 0 7 . 1 7 1 2 1 . 9 0 5 2 . 9 3 2 0 . 0 9 ( 7 . 1 6 ) ( 1 2 1 . 8 0 ) T 5 1 7 . 8 1 1 0 4 . 6 0 6 1 . 7 6 7 2 . 6 6 E 5 2 8 . 9 0 1 2 4 . 2 0 5 2 . 8 5 3 1 . 1 5 P 5 3 - - 6 4 . 5 7 3 1 . 8 1 S 5 4 7 . 3 1 1 1 2 . 5 0 5 6 . 2 0 6 4 . 7 9 Q 5 5 7 . 3 1 1 2 3 . 6 0 5 7 . 7 4 2 6 . 0 5 N 5 6 1 0 . 6 2 1 2 7 . 4 0 5 3 . 7 9 3 8 . 4 3 ( 1 0 . 6 0 ) ( 1 2 7 . 3 0 ) S 5 7 7 . 3 6 1 1 7 . 3 0 5 6 . 0 3 6 3 . 1 5 F 5 8 8 . 1 1 1 2 2 . 7 0 5 6 . 8 3 4 0 . 6 1 S 5 9 8 . 5 9 1 1 9 . 5 0 5 5 . 0 5 6 4 . 1 5 F 6 0 8 . 7 3 1 2 2 . 3 0 5 7 . 0 8 3 9 . 4 5 G 6 1 8 . 6 2 1 1 0 . 0 0 4 8 . 2 0 -A 6 2 8 . 7 5 1 2 5 . 3 0 5 5 . 3 8 1 7 . 7 4 G 6 3 9 . 1 0 1 0 9 . 3 0 4 7 . 4 1 D 6 4 9 . 9 3 1 2 4 . 7 0 5 7 . 3 8 3 9 . 1 1 R 6 5 7 . 9 0 1 2 2 . 1 0 5 9 . 2 0 2 9 . 5 1 V 6 6 7 . 5 1 1 2 1 . 1 0 6 6 . 3 5 . 3 0 . 2 2 A 6 7 8 . 4 3 1 2 2 . 0 0 5 5 . 1 4 1 7 . 6 2 S 6 8 8 . 9 7 1 1 7 . 1 0 6 1 . 6 9 6 2 . 7 8 Y 6 9 7 . 6 4 1 2 3 . 3 0 6 1 . 9 4 3 7 . 4 6 A 7 0 7 . 9 5 1 2 2 . 7 0 5 5 . 2 4 1 7 . 3 4 A 7 1 7 . 8 7 1 1 9 . 5 0 5 4 . 4 8 1 7 . 8 6 D 7 2 8 . 6 5 1 1 9 . 2 0 5 6 . 6 2 4 0 . 6 4 T 7 3 7 . 3 0 1 0 5 . 4 0 6 0 . 9 2 6 9 . 7 1 ( 7 . 3 3 ) ( 1 0 5 . 4 0 ) G 7 4 7 . 5 9 1 1 3 . 9 0 , 4 6 . 8 5 -K 7 5 8 . 4 1 1 2 0 . 6 0 5 5 . 0 2 3 6 . 0 0 E 7 6 7 . 7 4 1 2 1 . 7 0 5 6 . 0 9 3 0 . 7 3 L 7 7 8 . 7 6 1 2 5 . 0 0 5 3 . 0 0 4 2 . 9 1 ( 8 . 7 6 ) ( 1 2 5 . 1 0 ) Y 7 8 8 . 9 4 1 3 2 . 1 0 5 6 . 1 6 4 1 . 7 9 G 7 9 8 . 6 9 1 1 7 . 7 0 4 4 . 9 6 -H 8 0 8 . 0 1 1 2 9 . 1 0 5 1 . 7 8 3 2 . 4 3 T 8 1 6 . 3 6 1 2 0 . 2 0 5 9 . 9 4 7 0 . 7 1 L 8 2 7 . 7 2 1 2 9 . 3 0 5 8 . 7 6 4 0 . 7 3 C \ 1 7 6 . 8 0 C 5 . 1 8 2 . 3 0 C \ 1 7 6 . 5 0 H 5 , 7 . 9 9 ; N\ 3 5 . 8 9 H E 1 , 8 . 3 8 ; N B 1 , 1 2 5 . 5 0 C T , 1 7 9 . 2 0 C \ 3 2 . 1 0 ; C 6 , 1 7 9 . 1 0 ; H , ( 6 . 5 9 , 7 . 9 3 ) ; N B 2 , 1 1 0 . 3 0 C T ' 1 7 8 . 5 0 ; H 5 2 , ( 6 . 8 0 , 7 . 4 5 ) ; N 8 2 , 1 1 3 . 2 0 C\ 1 7 9 . 2 0 C 8 , 1 8 3 . 2 0 H 8 2 , 6 . 8 3 ; C 5 2 , 1 2 9 . 3 ; H B \ 6 . 6 4 ; C B \ 1 3 8 . 6 ; H 8 1 , 1 3 . 8 0 ; N 6 1 , 1 7 2 . 1 ; N B 2 , 2 4 6 . 9 Appendix 2 215 .esidue H N (40 °C) N H (40 °C) c° cp Other V 8 3 7 . 4 9 1 1 5 . 7 0 6 3 . 3 9 3 2 . 5 8 W84 6 . 7 6 1 2 4 . 3 0 5 3 . 3 4 - H E 1 , 1 0 . 0 8 ; N C \ 1 0 1 . 8 0 H 8 5 H 8 2 , 6 . 8 8 ; C 5 2 , 1 1 8 . 4 ; H E 1 , 8 . 4 3 ; C B \ 1 3 9 . 0 ; N 8 1 , 2 4 7 . 5 ; HP2, 1 1 . 8 0 ; N 8 2 , 1 6 7 . 5 S 8 6 Q 8 7 L 8 8 ^ P 8 9 - - 6 2 . 0 0 3 2 . 7 8 D 9 0 8 . 6 3 1 2 3 . 1 0 5 7 . 5 8 3 9 . 7 5 C \ 1 7 9 . 4 0 W 9 1 8 . 2 2 1 1 5 . 7 0 5 8 . 5 8 2 7 . 3 4 H B 1 , 9 . 5 3 ; N B 1 , 1 2 8 . 3 0 ( 8 . 1 8 ) ( 1 1 5 . 7 0 ) A 9 2 5 . 9 2 1 2 4 . 7 0 5 3 . 6 7 1 5 . 9 4 K 9 3 6 . 8 2 1 1 5 . 7 0 5 8 . 8 7 3 1 . 6 8 N 9 4 7 . 1 1 1 1 3 . 6 1 1 5 2 . 9 7 3 8 . 9 6 C, 1 7 6 . 8 0 ; H 8 2 , ( 6 . 8 2 , 7 . 3 4 ) ; N 6 2 , 1 1 3 . 6 0 L 9 5 7 . 0 4 1 2 0 . 4 0 5 3 . 9 2 4 3 . 4 6 ( 7 . 0 4 ) ( 1 2 0 . 3 0 ) C R , 1 7 6 . 8 0 ; H 6 2 , ( 6 . 8 6 , 7 . 5 2 ) ; N 5 2 , 1 1 5 . 8 0 N 9 6 8 . 5 9 1 1 8 . 1 11 5 1 . 8 3 4 2 . 6 3 G 9 7 8 . 8 8 1 0 8 . 8 0 4 6 . 8 7 -( 8 . 8 8 ) ( 1 0 8 . 8 0 ) S 9 8 - - 6 1 . 0 3 6 2 . 6 9 A 9 9 8 . 3 0 1 2 5 . 8 0 5 4 . 7 7 1 8 . 1 9 F 1 0 0 7 . 5 3 1 2 1 . 8 0 6 0 . 5 3 3 7 . 5 5 E 1 0 1 8 . 4 6 1 2 1 . 5 0 6 0 . 5 2 2 9 . 7 7 C 5 , 1 8 2 . 8 0 S 1 0 2 8 . 7 6 1 1 3 . 9 0 6 1 . 4 5 6 2 . 7 1 ( 8 . 7 4 ) ( 1 1 3 . 9 0 ) A 1 0 3 7 . 8 8 1 2 4 . 9 0 5 4 . 7 7 1 8 . 0 6 M 1 0 4 8 . 3 1 1 2 0 . 8 0 5 9 . 4 8 3 1 . 9 6 V 1 0 5 8 . 7 6 1 2 0 . 8 0 6 7 . 6 1 3 1 . 2 9 N 1 0 6 8 . 9 7 1 2 1 . 6 0 5 7 . 0 0 3 9 . 1 5 C \ 1 7 6 . 1 0 H 1 0 7 8 . 3 4 1 1 8 . 6 0 6 1 . 0 2 2 8 . 3 8 H 8 2 , 6 . 3 6 ; C 8 2 , 1 1 8 . 8 ; H B \ 8 . 3 0 ; C B \ 1 3 6 . 0 ; H 8 1 , 1 6 . 6 8 ; N 8 1 , 1 8 4 . 6 ; H C 2 , 1 3 . 5 0 ; NP2, 1 7 0 . 6 V 1 0 8 8 . 1 6 1 1 6 . 3 0 6 6 . 1 5 3 1 . 4 9 T 1 0 9 8 . 1 0 1 1 2 . 6 0 6 6 . 2 5 6 8 . 9 8 K 1 1 0 8 . 2 3 1 1 8 . 7 0 5 8 . 2 6 3 1 . 2 0 V 1 1 1 7 . 7 3 1 2 0 . 0 0 - 3 1 . 5 5 A 1 1 2 7 . 7 5 1 2 0 . 6 0 5 4 . 9 9 1 6 . 3 8 D 1 1 3 8 . 6 2 1 1 7 . 0 0 5 7 . 2 5 4 2 . 5 6 C \ 1 7 9 . 1 0 H 1 1 4 7 . 4 2 1 1 6 . 3 0 5 9 . 2 7 2 7 . 8 1 H 8 2 , 3 . 7 6 ; C 8 2 , 1 1 8 . 8 ; HP1, 8 . 2 3 ; C B \ 1 3 5 . 7 ; N 6 1 , ( 7 . 4 4 ) ( 1 1 6 . 4 0 ) 1 8 1 . 4 ; NP2, 1 7 7 . 4 F 1 1 5 7 . 1 4 1 1 2 . 5 0 5 8 . 2 4 3 9 . 9 1 E 1 1 6 7 . 2 2 1 2 6 . 1 0 5 8 . 4 7 2 7 . 9 7 C 6 , 1 8 2 . 3 0 G 1 1 7 9 . 7 0 ' 1 1 8 . 6 0 4 5 . 0 8 -( 9 . 6 8 ) ( 1 1 8 . 6 0 ) K 1 1 8 8 . 2 4 1 1 9 . 3 0 5 6 . 8 0 3 3 . 0 5 V 1 1 9 7 . 3 4 1 1 7 . 6 0 6 0 . 1 3 3 6 . 2 4 A 1 2 0 8 . 3 5 1 2 8 . 6 0 5 3 . 1 3 1 9 . 8 1 S 1 2 1 6 . 5 9 1 1 1 . 3 0 5 5 . 9 6 6 6 . 9 8 W 1 2 2 8 . 9 4 1 2 1 . 6 0 5 5 . 5 8 3 4 . 9 2 H B \ 1 2 . 0 2 ; N B \ 1 0 1 . 5 0 D 1 2 3 8 . 7 2 1 1 9 . 9 0 5 3 . 5 9 4 0 . 3 6 V 1 2 4 9 . 4 7 1 3 2 . 7 0 6 5 . 4 6 3 0 . 3 9 ( 9 . 4 7 ) ( 1 3 2 . 5 0 ) V 1 2 5 6 . 4 3 1 1 2 . 2 0 6 0 . 3 3 3 5 . 3 3 N 1 2 6 8 . 3 5 1 2 6 . 4 0 5 0 . 4 1 4 1 . 5 2 C , 1 7 4 . 2 0 E 1 2 7 8 . 8 2 1 2 1 . 2 0 5 7 . 0 0 2 6 . 5 2 C 8 , 1 8 3 . 0 0 ( 8 . 8 4 ) ( 1 2 1 . 2 0 ) Appendix 2 216 Residue H N N H C a C" (40 °C) (40 °C) A128 8.88 118.40 52.95 19.95 (8.87) (118.5) F129 8.10 117.60 59.22 40.23 A130 8.67 124.00 49.88 20.26 D131 8.58 122.90 57.15 39.88 G132 - - 44.72 -G133 7.93 108.80 43.55 -G134 8.28 106.70 43.96 -R135 8.68 116.50 53.49 30.35 R136 8.00 122.60 57.10 29.39 Q137 8.64 124.10 56.09 28.58 D138 8.20 118.70 52.27 38.24 S139 6.51 111.80 54.13 63.77 (6.51) (111.70) A140 8.98 132.30 54.53 17.79 F141 6.97 113.40 61.21 36.72 Q142 6.80 119.80 57.25 29.33 Q143 8.34 116.60 58.40 28.28 (8.34) (116.50) K144 7.82 114.80 53.83 -(7.83) (114.70) L145 7.84 114.90 - -G146 7.59 109.80 - -N147 8.73 115.40 -• -G148 - - 46.56 -Y149 7.14 117.50 60.04 38.60 1150 5.87 120.20 65.54 36.29 E151 8.74 122.80 59.83 27.86 T152 7.84 116.40 66.01 67.68 (7.86) (116.40) A153 8.39 124.40 55.98 17.53 F154 8.36 117.10 63.95 38.40 R155 8.51 117.00 60.42 28.69 A156 8.09 122.30 54.13 17.42 A157 8.18 123.20 55.06 17.87 R158 7.82 117.10 55.87 27.37 A159 7.35 119.30 53.60 17.53 A160 7.11 119.20 52.70 21.09 D161 7.78 114.80 50.55 41.13 P162 - - 63.67 32.60 T163 8.25 110.50 62.25 70.07 A164 7.61 126.90 52.09 18.23 K165 8.46 122.50 54.94 31.07 L166 10.44 125.20 54.17 40.60 C167 10.03 123.10 52.13 39.29 1168 8.10 121.90 58.68 41.73 N169 8.03 126.30 52.03 43.22 D170 8.50 127.50 53.89 46.35 Y171 10.12 118.20 54.52 40.51 (10.15) (118.30) N172 9.17 121.80 54.19 35.95 V173 6.57 103.90 58.88 31.03 (6.59) (103.90) C8, 180.30 C J, 180.70 C6, 180.10 C\ 33.12; C8, 180.00; hf2, (6.62, 7.32); NT, 111.10 C8, 182.40 Cy, 180.60 Appendix 2 2 1 7 esidue H N (40 °C) N H (40 °C) C " c p Other E174 8.12 121.10 61.71 27.27 C 6, 183.80 G175 7.43 103.10 42.50 -1176 (7.43) 8.23 (103.10) 120.00 63.07 34.92 N177 7.90 127.20 51.63 38.51 A178 ' - - 55.34 18.19 K179 8.22 121.90 60.54 33.15 S180 9.51 116.30 60.82 63.05 N181 9.00 123.70 55.53 36.26 C\ 175.80 S182 7.86 119.80 62.64 63.50 L183 (7.86) 7.41 (119.80) 126.40 57.53 41.18 Y184 8.92 122.30 61.79 37.53 D185 7.97 118.70 57.01 40.09 C , 179.00 L186 7.32 122.80 58.34 40.73 V187 (7.35) 8.18 (122.70) 121.20 66.64 30.20 K188 8.45 119.60 59.91 31.98 D189 7.66 120.30 57.01 42.35 C',180.20 F190 9.20 121.00 59.53 36.85 K191 (9.19) 8.65 (120.90) 116.60 56.10 28.55 A192 8.09 123.80 54.72 17.96 R193 7.81 114.20 56.47 30.96 G194 7.65 109.40 45.96 V195 8.34 125.40 60.31 31.79 P196 - - 63.60 30.81 L197 7.29 121.70 56.95 42.07 D198 9.81 123.60 54.70 45.06 C199 8.13 116.60 53.37 49.19 V200 (8.16) 8.56 (116.50) 120.80 60.56 34.41 G201 9.54 115.60 44.36 -F202 8.46 122.40 - -Q203 9.01 125.20 59.67 27.73 C s , 179.60 S204 8.70 102.60 58.40 61.75 H205 (8.75) 8.31 (102.70) 118.10 54.54 27.25 H 5 2, 7.23; C 6 2 , 119.5; H c \ 7.75; C s 1 , 136.4; 17.64; N s 1, 188.0; HP2, 10.22; NP2, 164.3 L206 8.62 125.50 52.60 43.95 I207 8.39 124.10 60.01 40.26 V208 8.19 125.00 62.43 31.63 C r, (21.31,22.80) G209 11.92 119.80 44.55 . -Q210 8.37 120.80 53.62 27.90 CY, 32.87; C s, 180.60; HP2, (6.67, 7.32); NP2, 112.30 V211 8.84 126.70 61.69 31.50 P212 (8.77) (126.60) 63.42 31.33 G213 9.00 113.70 46.27 -D214 8.98 118.70 51.81 38.09 C\ 180.60 F215 6.94 121.50 58.04 39.01 R216 (6.99) 8.94 (121.50) 118.90 60.33 28.08 Q217 8.45 118.90 59.15 27.52 C\ 33.65; C 6, 180.10; FP2, (6.82, 7.46); NP2, 112.30 Appendix 2 218 esidue H N N H c° C (40 °C) (40 °C) N218 8.55 120.20 55.71 38.98 (8.56) (120.20) L219 8.61 119.00 - -Q220 8.69 117.10 57.46 28.68 R221 8.06 117.60 58.65 26.88 (8.08) (117.50) F222 7.61 121.80 . 63.05 39.46 A223 8.28 123.80 54.15 18.32 D224 8.57 118.70 56.30 40.27 L225 7.88 120.20 55.24 41.79 (7.89) (120.10) G226 8.41 106.60 45.23 • -V227 6.85 110.80 57.96 34.00 D228 7.64 117.60 53.55 42.32 V229 8.14 110.20 57.84 35.11 R230 8.67 120.90 55.80 34.35 1231 8.03 128.30 60.38 38.06 T232 8.04 117.50 63.32 69.51 E233 7.71 123.70 52.66 30.73 L234 7.92 115.80 55.85 41.53 (7.92) (115.80) D235 8.30 119.70 52.75 41.38 I236 8.11 119.20 60.11 37.77 (8.12) (119.40) R237 8.43 123.00 54.77 32.43 M238 8.36 117.20 52.46 37.15 R239 8.51 121.00 55.77 28.76 T240 8.00 117.60 - -P241 S242 D243 A244 - - 55.69 18.24 T245 8.21 117.80 66.12 68.19 K246 9.00 125.60 60.12 34.72 L247 8.45 117.20 57.63 41.59 A248 7.93 125.00 54.81 17.60 T249 8.43 120.80 66.39 68.21 Q250 8.61 120.20 58.86 30.30 A251 7.91 122.20 55.79 17.45 A252 7.53 120.80 54.62 17.45 D253 8.52 123.10 57.62 41.26 Y254 8.59 121.20 63.80 38.21 K255 K256 - - 59.50 32.69 V257 7.95 120.60 66.55 31.27 V258 7.87 118.30 66.81 29.81 Q259 8.84 119.00 59.37 28.32 A260 7.57 121.70 55.26 16.35 (7.61) (121.70) C261 6.91 111.50 57.55 41.36 M262 8.41 118.50 55.75 30.04 Q263 7.58 116.90 56.12 28.90 C8, 180.30 C\ 179.20 C\ 178.80 C8, 174.50 C, 181.10 C, 175.80 C r, 34.78; C5, 180.30; HP2, (6.76); NP2, 112.50 C\ 34.16; C8, 180.30; HP2, (6.60, 7.16); Appendix 2 219 Residue H N (40 °C) N H (40 °C) C" c p Other V 2 6 4 7 . 6 4 1 2 4 . 2 0 6 1 . 1 4 3 2 . 6 8 T 2 6 5 ( 7 . 6 5 ) 9 . 1 1 ( 1 2 4 . 1 0 ) 1 2 4 . 0 0 6 6 . 0 2 6 8 . 3 2 R 2 6 6 7 . 2 8 1 1 4 . 3 0 5 5 . 9 5 2 9 . 2 2 C 2 6 7 8 . 1 8 1 2 2 . 4 0 5 5 . 5 1 4 4 . 6 9 Q 2 6 8 9 . 2 6 1 2 8 . 6 0 5 6 . 1 0 3 0 . 0 2 C T , 3 3 . 6 9 ; C 5 , 1 7 8 . 4 0 ; N" 2 , G 2 6 9 7 . 3 3 1 0 1 . 5 0 4 5 . 6 0 -V 2 7 0 ( 7 . 3 5 ) 8 . 3 4 ( 1 0 1 . 4 0 ) 1 1 8 . 6 0 6 0 . 2 7 3 6 . 7 1 T 2 7 1 8 . 6 2 1 2 7 . 8 0 5 9 . 8 4 7 0 . 1 8 V 2 7 2 8 . 3 6 1 2 0 . 0 0 5 8 . 5 4 3 3 . 3 8 W 2 7 3 9 . 0 5 1 2 9 . 7 0 5 5 . 7 1 2 7 . 1 5 H S 1 , 1 0 0 . 2 0 ; N C \ 1 1 4 . 2 0 G 2 7 4 8 . 2 1 1 1 2 . 1 0 4 2 . 8 0 -I 2 7 5 8 . 8 1 1 1 6 . 0 0 6 3 . 4 4 4 0 . 5 5 T 2 7 6 7 . 1 7 1 1 0 . 0 0 5 6 . 0 8 6 9 . 5 1 D 2 7 7 8 . 3 8 1 3 0 . 9 0 5 6 . 9 4 4 1 . 9 6 C \ 1 7 6 . 0 0 K 2 7 8 7 . 7 7 1 1 9 . 0 0 5 8 . 9 0 3 3 . 3 4 Y 2 7 9 ( 7 . 7 6 ) 7 . 2 4 ( 1 1 8 . 8 0 ) 1 1 5 . 9 0 5 4 . 3 9 3 8 . 6 3 S 2 8 0 7 . 1 6 1 1 4 . 0 0 5 7 . 0 1 6 4 . 1 3 W 2 8 1 8 . 3 0 1 2 8 . 5 0 5 8 . 4 5 2 8 . 1 0 H S 1 , 1 2 0 . 1 5 ; NT1, 1 0 1 . 9 0 V 2 8 2 6 . 7 6 1 1 9 . 8 0 - -P 2 8 3 - - 6 4 . 6 9 3 0 . 5 3 D 2 8 4 7 . 2 5 1 1 5 . 5 0 5 5 . 8 8 4 0 . 6 1 C R , 1 7 9 . 8 0 V 2 8 5 7 . 2 3 1 2 0 . 0 0 6 3 . 5 0 3 3 . 6 7 F 2 8 6 ( 7 . 2 6 ) 8 . 3 9 ( 1 2 0 . 0 0 ) 1 2 1 . 3 0 5 4 . 2 4 3 8 . 9 8 P 2 8 7 - - 6 3 . 4 5 3 1 . 0 0 G 2 8 8 8 . 7 9 1 1 2 . 0 0 4 4 . 7 9 -E 2 8 9 8 . 2 9 1 2 0 . 1 0 5 3 . 9 4 3 3 . 0 5 C 5 , 1 8 2 . 5 0 G 2 9 0 8 . 7 1 1 0 4 . 7 0 4 6 . 2 4 -A 2 9 1 ( 8 . 6 9 ) 8 . 2 2 ( 1 0 4 . 4 0 ) 1 2 4 . 5 0 5 2 . 0 4 1 7 . 7 2 A 2 9 2 8 . 7 6 1 2 0 . 7 0 5 3 . 7 3 2 2 . 5 2 L 2 9 3 9 . 4 5 1 2 3 . 6 0 5 4 . 3 1 4 1 . 6 0 V 2 9 4 8 . 0 2 1 1 9 . 4 0 6 5 . 4 4 3 0 . 3 2 W 2 9 5 7 . 3 4 1 1 6 . 6 0 5 8 . 0 6 3 2 . 5 3 H E 1 , 1 0 0 . 0 9 ; N B 1 , 1 3 0 . 5 0 D 2 9 6 8 . 7 4 1 2 0 . 0 0 - -A 2 9 7 - - 5 4 . 1 9 1 7 . 9 0 S 2 9 8 8 . 4 5 1 1 4 . 5 0 5 7 . 3 4 6 3 . 8 9 Y 2 9 9 7 . 9 4 1 1 3 . 7 0 6 1 . 9 0 3 3 . 4 1 A 3 0 0 8 . 4 8 1 2 3 . 6 0 5 0 . 8 8 1 8 . 8 3 K 3 0 1 8 . 4 4 1 2 2 . 1 0 5 7 . 2 5 3 2 . 7 5 K 3 0 2 7 . 7 0 1 2 5 . 7 0 5 5 . 0 2 2 9 . 6 2 H\ 8 . 0 7 ; N 5 , 3 5 . 1 8 P 3 0 3 - -' 6 5 . 3 8 3 0 . 5 7 A 3 0 4 7 . 6 2 1 1 7 . 3 0 5 4 . 3 9 1 9 . 3 8 Y 3 0 5 7 . 8 9 1 1 8 . 4 0 6 2 . 4 4 3 7 . 6 8 A 3 0 6 7 . 6 2 1 2 0 . 3 0 5 3 . 9 1 1 7 . 9 2 A 3 0 7 6 . 9 7 1 2 0 . 6 0 5 4 . 4 8 2 0 . 2 9 V 3 0 8 7 . 8 2 1 1 9 . 6 0 6 6 . 7 1 3 0 . 4 2 M 3 0 9 7 . 4 7 1 1 8 . 8 0 5 9 . 1 7 3 1 . 8 4 E 3 1 0 8 . 0 6 1 1 7 . 9 0 5 8 . 1 8 2 9 . 4 3 C 5 , 1 8 3 . 4 0 Appendix 2 220 Residue H N N H C a C p Other (40 °C) (40 °C) A311 8.12 124.10 54.34 19.73 (8.13) (124.10) F312 7.58 115.00 59.29 39.52 G313 7.84 107.00 45.09 -A314 7.48 123.60 51.12 19.81 S315 8.12 116.70 55.89 63.73 Appendix 3 221 Append ix 3 N M R R e l a x a t i o n P a r a m e t e r s f o r a p o - C e x C D 3 Residue Ti (sec) T 2(sec) NOE s 2 A1 T2 T3 1.22 + 0.04 0.058 ± 0.002 0.71 ± 0.04 0.82 ± 0.05 L4 K5 1.31 + 0.05 0.051 + 0.001 0.76 + 0.04 0.87 ± 0.05 E6 1.27 ± 0.04 0.047 + 0.001 0.86 + 0.04 0.92 + 0.04 . A7 A8 1.23 + 0.04 0.052 ± 0.001 0.88 + 0.04 0.89 + 0.05 D9 1.22 ± 0.04 0.048 + 0.001 0.80 + 0.04 0.92 + 0.05 G10 1.21 + 0.04 0.050 + 0.001 0.87 + 0.04 0.90 + 0.04 A11 1.26 + 0.04 0.052 ± 0.001 0.84 + 0.04 0.87 ± 0.05 G12 1.30 + 0.04 0.054 ± 0.002 0.82 + 0.04 0.84 ± 0.04 R13 1.39 + 0.05 0.046 ± 0.001 0.87 + 0.04 0.90 + 0.05 D14 1.29 ± 0.04 0.049 ± 0.001 0.83 ± 0.04 0.90 ± 0.05 F15 1.26 + 0.04 0.053 + 0.002 0.83 + 0.04 0.87 + 0.05 G16 F17 A18 1.38 ± 0.05 0.045 ± 0.001 0.69 + 0.03 0.80 + 0.07 L19 D20 P21 N22 1.08 ± 0.04 0.048 ± 0.001 0.72 ± 0.04 0.93 ± 0.03 R23 1.34 ± 0.05 0.044 ± 0.001 0.89 + 0.04 0.94 ± 0.04 L24 S25 1.30 + 0.05 0.045 ± 0.001 0.74 ± 0.04 0.93 ± 0.04 E26 1.31 + 0.04 0.052 ± 0.001 0.74 ± 0.04 0.86 ± 0.05 A27 Q28 1.17 ± 0.04 0.046 ± 0.001 0.78 ± 0.04 0.97 ± 0.04 Y29 1.29 ± 0.05 0.046 + 0.001 0.73 + 0.04 0.92 + 0.04 K30 1.21 + 0.04 0.045 + 0.001 0.84 + 0.04 0.97 + 0.04 A31 1.21 + 0.04 0.047 ± 0.001 0.86 ± 0.04 0.95 + 0.04 I32 1.23 ± 0.04 0.049 + 0.001 0.86 ± 0.04 0.92 ± 0.05 A33 D34 1.24 ± 0.04 0.053 ± 0.002 0.74 + 0.04 0.86 ± 0.05 S35 E36 1.23 ± 0.04 0.046 + 0.001 0.85 + 0.04 0.95 + 0.04 F37 1.29 + 0.04 0.054 ± 0.002 0.87 + 0.04 0.85 ± 0.05 N38 1.26 + 0.04 0.049 ± 0.001 0.88 ± 0.04 0.91 + 0.04 L39 V40 1.22 0.04 0.045 + 0.001 0.86 + 0.04 0.96 + 0.04 V41 • A42 E43 N44 A45 3 List of the relaxation parameters determined for apo-CexCD at pH 6.5 and 30 °C. Appendix 3 222 Residue M46 K47 W48 W48-lndole D49 A50 T51 E52 P53 S54 Q55 N56 S57 F58 S59 F60 G61 A62 G63 D64 R65 V66 A67 S68 Y69 A70 A71 D72 T73 G74 K75 E76 L77 Y78 G79 H80 T81 L82 V83 W84 W84-lndole H85 S86 Q87 L88 P89 D90 W91 W91-lndole A92 K93 N94 L95 Ti(sec) 1.34 + 0.05 1.33 + 0.05 1.33 ± 0.05 1.60 + 0.11 1.22 + 0.04 1.30 + 0.04 1.25 ± 0.04 1.26 + 0.04 1.30 + 0.05 1.28 ± 0.04 1.27 + 0.04 1.39 + 0.05 1.31 + 0.04 1.35 ± 0.05 1.31 + 0.05 1.35 + 0.05 1.32 ± 0.05 1.26 + 0.04 T 2(sec) 0.049 ± 0.001 0.048 + 0.001 0.047 ± 0.001 0.057 ± 0.002 0.042 + 0.001 0.053 ± 0.001 0.050 + 0.001 0.052 ± 0.002 0.052 + 0.001 0.050 ± 0.001 0.050 ± 0.001 0.056 ± 0.002 0.052 ± 0.001 0.049 + 0.001 0.046 ± 0.001 0.048 ± 0.001 0.047 ± 0.001 0.045 ± 0.001 N O E 0.83 + 0.04 0.79 ± 0.04 0.84 ± 0.04 0.75 0.04 0.86 + 0.04 0.80 ± 0.04 0.82 + 0.04 0.83 + 0.04 0.79 + 0.04 0.81 + 0.04 0.83 + 0.04 0.76 + 0.04 0.78 ± 0.04 0.82 + 0.04 0.80 ± 0.04 0.81 ± 0.04 0.82 ± 0.04 0.84 + 0.04 s2 0.88 ± 0.04 0.89 ± 0.05 0.92 ± 0.05 0.86 + 0.01 1.00 ± 0.03 0.86 ± 0.05 0.90 + 0.05 0.88 ± 0.05 0.87 + 0.05 0.89 ± 0.05 0.90 ± 0.04 0.80 ± 0.05 0.86 ± 0.05 0.88 ± 0.05 0.93 ± 0.05 0.88 ± 0.05 0.91 ± 0.05 0.95 + 0.04 1.28 + 0.04 1.28 ± 0.04 1.33 + 0.05 1.32 + 0.05 1.40 ± 0.05 1.37 ± 0.05 1.33 ± 0.05 1.39 ± 0.05 1.29 0.04 1.23 + 0.04 1.29 + 0.04 1.35 + 0.05 1.40 + 0.05 1.36 ± 0.05 1.52 + 0.19 1.31 + 0.04 1.22 + 0.04 1.56 ± 0.12 1.31 ± 0.04 1.24 ± 0.04 1.21 + 0.04 0.046 ± 0.001 0.044 + 0.001 0.047 ± 0.001 0.047 + 0.001 0.047 ± 0.001 0.050 + 0.001 0.049 0.001 0.046 ± 0.001 0.054 ± 0.002 0.042 + 0.001 0.048 + 0.001 0.043 ± 0.001 0.046 ± 0.001 0.048 ± 0.001 0.053 + 0.004 0.050 ± 0.001 0.048 ± 0.001 0.051 ± 0.002 0.048 ± 0.001 0.049 ± 0.001 0.054 ± 0.002 0.81 ± 0.04 0.86 ± 0.04 0.85 ± 0.04 0.82 + 0.04 0.86 + 0.04 0.73 + 0.04 0.86 ± 0.04 0.83 ± 0.04 0.76 + 0.04 0.72 + 0.04 0.84 ± 0.04 0.86 ± 0.04 0.84 ± 0.04 0.83 ± 0.04 0.77 + 0.04 0.85 ± 0.04 0.82 + 0.04 0.81 ± 0.04 0.75 ± 0.04 0.85 ± 0.04 0.78 ± 0.04 0.94 + 0.05 0.96 ± 0.04 0.90 ± 0.04 0.91 + 0.05 0.89 + 0.05 0.85 + 0.05 0.88 ± 0.04 0.90 ± 0.05 0.84 ± 0.05 0.97 + 0.03 0.91 ± 0.04 0.95 ± 0.04 0.90 ± 0.05 0.89 + 0.04 0.92 + 0.03 0.88 + 0.05 0.92 + 0.04 Q.92 ± 0.02 0.90 ± 0.05 0.90 + 0.05 0.86 ± 0.04 Appendix 3 223 Residue Ti (sec) T 2(sec) NOE s2 N96 1.29 ± 0.04 . 0.050 + 0.001 0.73 ± 0.04 0.87 + 0.04 G97 1.17 ± 0.04 0.059 ± 0.002 0.69 + 0.03 0.82 + 0.05 S98 A99 1.20 + 0.04 0.051 + 0.001 0.82 + 0.04 0.89 + 0.05 F100 E101 1.29 + 0.04 0.044 ± 0.001 0.83 + 0.04 0.95 + 0.04 S102 1.31 + 0.05 0.046 ± 0.001 0.89 ± 0.04 0.92 + 0.05 A103 M104 1.32 ± 0.05 0.046 + 0.001 0.87 ± 0.04 0.92 ± 0.05 V105 N106 1.26 ± 0.04 0.045 ± 0.001 0.87 ± 0.04 0.95 + 0.04 H107 V108 T109 K110 V111 A112 D113 1.31 ± 0.05 0.043 ± 0.001 0.82 ± 0.04 0.96 + 0.04 H114 1.28 ± 0.04 0.046 ± 0.001 0.74 ± 0.04 0.93 + 0.04 F115 1.31 ± 0.04 0.050 + 0.001 0.84 ± 0.04 0.88 ± 0.04 E116 1.37 + 0.05 0.045 ± 0.001 0.82 + 0.04 0.91 + 0.05 G117 1.29 ± 0.04 0.051 ± 0.001 0.84 ± 0.04 0.88 + 0.05 . K118 V119 A120 1.32 ± 0.05 0.046 ± 0.001 0.84 ± 0.04 0.91 ± 0.05 S121 1.34 + 0.05 0.056 + 0.002 0.82 ± 0.04 0.82 ± 0.05 W122 W122-lndole 1.42 + 0.07 0.054 + 0.005 0.82 ± 0.04 0.93 ± 0.02 D123 1.39 ± 0.05 0.051 ± 0.001 0.87 ± 0.04 0.85 + 0.05 V124 V125 N126 E127 A128 1.26 ± 0.04 0.045 ± 0.001 0.75 ±. 0.04 0.95 ± 0.04 F129 1.28 ± 0.04 0.044 + 0.001 0.85 + 0.04 0.96 ± 0.04 A130 1.28 ± 0.05 0.047 ± 0.001 0.77 ± 0.04 0.92 + 0.05 D131 G132 G133 1.22 ± 0.04 0.050 ± 0.001 0.79 + 0.04 0.90 + 0.05 G134 1.00 ± 0.03 0.058 ± 0.002 0.87 + ' 0.04 0.70 + 0.10 R135 1.27 0.04 0.048 + 0.001 0.84 ± 0.04 0.91 + 0.05 R136 1.21 0.04 0.053 + 0.002 0.82 ± 0.04 0.88 ± 0.05 Q137 1.31 ± 0.05 0.051 ± 0.001 0.76 ± 0.04 0.86 ± 0.04 D138 1.21 ± 0.04 0.047 ± 0.001 0.75 + 0.04 0.92 + 0.04 S139 c A140 1.18 ± 0.04 0.043 ± 0.001 0.74 ± 0.04 0.98 ± 0.03 F141 1.36 ± 0.05 0.045 ± 0.001 0.84 ± 0.04 0.91 ± 0.05 Q142 1.40 ± 0.05 0.045 + 0.001 0.78 + 0.04 0.91 ± 0.05 Q143 1.29 ± 0.04 0.043 ± 0.001 0.85 + 0.04 0.97 ± 0.04 K144 L145 1.25 ± 0.04 0.045 + 0.001 0.79 ± 0.04 0.95 ± 0.04 G146 1.30 ± 0.05 0.051 ± 0.001 0.89 + 0.04 0.88 ± 0.05 N147 Appendix 3 224 Residue Ti (sec) T 2(sec) NOE s2 G148 Y149 1.14 + 0.04 0.046 + 0.001 0.83 ± 0.04 0.98 ± 0.04 1150 1.18 + 0.04 0.045 + 0.001 0.82 ± 0.04 0.99 + 0.03 E151 1.22 + 0.04 0.051 + 0.001 0.80 ± 0.04 0.90 ± 0.05 T152 A153 1.06 ± 0.03 0.049 ± 0.001 0.88 ± 0.04 0.98 + 0.04 F154 1.26 ± 0.04 0.043 •+ 0.001 0.79 + 0.04 0.98 + 0.04 R155 1.26 + 0.04 0.048 + 0.001 0.82 + 0.04 0.92 + 0.05 A156 1.19 + 0.04 0.048 + 0.001 0.89 + 0.04 0.95 + 0.04 A157 1.15 ± 0.04 0.053 + 0.002 0.80 + 0.04 0.91 + 0.05 R158 1.24 ± 0.04 0.048 ± 0.001 0.75 ± 0.04 0.93 ± 0.05 A159 A160 D161 1.35 + 0.05 0.050 + 0.001 0.77 + 0.04 0.87 + 0.04 P162 T163 1.27 ± 0.04 0.056 + 0.002 0.76 + 0.04 0.83 ± 0.05 A164 1.35 + 0.05 0.052 + 0.001 0.81 + 0.04 0.84 + 0.05 K165 1.33 ± 0.05 0.049 + 0.001 0.89 ± 0.04 0.88 ± 0.05 L166 1.31 + 0.05 0.052 + 0.001 0.81 + 0.04 0.86 ± 0.04 C167 1168 1.13 ± 0.04 0.047 + 0.001 0.74 + 0.04 0.97 + 0.04 N169 D170 Y171 N172 1.28 ± 0.04 0.046 + 0.001 0.80 + 0.04 0.93 + 0.05 ' V173 1.28 + 0.04 0.047 ± 0.001 0.84 + 0.04 0.92 + 0.05 E174 1.26 + 0.04 0.061 + 0.002 0.71 + 0.04 0.79 + 0.05 G175 1.34 ± 0.05 0.054 + 0.002 0.86 + 0.04 0.83 + 0.05 1176 N177 1.34 + 0.05 0.054 ± 0.002 0.77 ± 0.04 0.83 ± 0.04 A178 K179 1.31 + 0.05 0.047 ± 0.001 0.81 + 0.04 0.90 + 0.05 S180 1.23 + 0.04 0.046 ± 0.001 0.80 + 0.04 0.95 + 0.05 N181 1.18 + 0.04 0.050 + 0.001 0.85 + 0.04 0.93 + 0.05 S182 L183 Y184 1.15 + 0.04 0.046 + 0.001 0.73 + 0.04 0.97 + 0.03 D185 1.21 ± 0.04 0.044 ± 0.001 0.77 ± 0.04 0.98 ± 0.03 L186 1.30 ± 0.04 0.050 + 0.001 0.88 + 0.04 0.89 + 0.05 V187 1.17 ± 0.04 0.048 ± 0.001 0.80 ± 0.04 0.95 + 0.05 K188 1.17 + 0.04 0.046 + 0.001 0.87 + 0.04 0.97 + 0.04 D189 F190 1.28 ± 0.04 0.047 + 0.001 0:80 + 0.04 0.93 ± 0.04 K191 1.28 ± 0.04 0.048 ± 0.001 0.85 ± 0.04 0.91 + 0.04 A192 1.20 ± 0.04 0.053 ± 0.002 0.87 + 0.04 0.88 ± 0.05 R193 1.36 ± 0.05 0.052 ± 0.001 0.77 ± 0.04 0.85 ± 0.05 G194 V195 P196 L197 D198 1.25 + 0.04 0.052 + 0.001 0.81 + 0.04 0.88 + 0.05 C199 V200 Appendix 3 225 Residue Ti (sec) T 2(sec) NOE s2 G201 1.18 ± 0.04 0.042 + 0.001 0.73 ± 0.04 0.98 ± 0.03 F202 Q203 S204 1.34 + 0.05 0.044 + 0.001 0.87 + 0.04 0.93 + 0.04 . H205 L206 1.39 + 0.05 0.053 + 0.002 0.76 ± 0.04 0.83 + 0.05 I207 1.29 ± 0.04 0.054 ± 0.002 0.82 ± 0.04 0.85 + 0.05 V208 G209 i .32 ± 0.05 0.052 ± 0.001 0.80 ± 0.04 0.86 ± 0.05 Q210 1.24 + 0.04 0.052 ± 0.001 0.77 ± 0.04 0.89 + 0.05 V211 1.40 ± 0.05 0.048 + 0.001 0.81 ± 0.04 0.87 + 0.05 P212 G213 1.32 + 0.05 0.048 ± 0.001 0.86 + 0.04 0.90 + 0.05 D214 1.12 + 0.04 0.047 + 0.001 0.82 ± 0.04 0.98 + 0.04 F215 1.29 + 0.04 0.046 + 0.001 0.84 + 0.04 0.93 + 0.04 R216 1.23 + 0.04 0.044 ± 0.001 0.85 + 0.04 0.96 + 0.04 Q217 1.31 ± 0.05 0.048 ± 0.001 0.80 ± 0.04 0.91 + 0.05 N218 1.28 ± 0.04 0.045 ± 0.001 0.84 + 0.04 0.95 ± 0.05 L219 Q220 1.32 + 0.05 0.042 ± 0.001 0.80 ± 0.04 0.97 + 0.04 R221 1.20 + 0.04 0.043 ± 0.001 0.85 ± 0.04 1.00 + 0.03 F222 1.32 ± 0.04 0.042 ± 0.001 . 0.82 + 0.04 0.96 ± 0.04 A223 1.25 ± 0.04 0.052 ± 0.002 0.88 + 0.04 0.86 ± 0.05 D224 1.23 + 0.04 • 0.044 + 0.001 0.84 + 0.04 0.98 + 0.03 L225 1.28 + 0.04 0.048 ± 0.001 0.85 + 0.04 0.91 ± 0.04 G226 1.38 + 0.05 0.048 + 0.001 0.81 + 0.04 0.88 0.05 V227 1.32 ± 0.05 0.052 ± 0.001 0.84 + 0.04 0.85 + 0.05 D228 V229 R230 1.12 + 0.04 0.041 + 0.001 0.87 + 0.04 1.00 ± 0.03 1231 1.17 + 0.04 0.049 ± 0.001 0.84 + 0.04 0.93 + 0.04 T232 1.18 + 0.04 0.039 ± 0.001 0.84 + 0.04 1.00 ± 0.03 E233 L234 D235 I236 R237 M238 1.17 + 0.04 0.047 ± 0.001 0.88 + 0.04 0.96 + 0.04 R239 1.26 + 0.04 0.050 ± 0.001 0.79 ± 0.04 0.89 ± 0.05 T240 1.38 + 0.05 0.047 ± 0.001 0.86 ± 0.04 0.89 ± 0.05 P241 S242 D243 A244 T245 K246 1.23 ± 0.04 0.052 + 0.001 0.80 + 0.04 0.88 + 0.05 1247 1.25 ± 0.04 0.047 ± 0.001 0.84 ± 0.04 0.93 ± 0.05 A248 1.19 + 0.04 0.048 ± 0.001 0.74 + 0.04 0.94 + 0.04 T249 1.22 ± 0.04 0.048 ± 0.001 0.85 + 0.04 0.94 + 0.04 Q250 1.28 + 0.04 0.045 ± 0.001 0.85 + 0.04 0.94 ± 0.05 A251 A252 1.26 + 0.04 0.048 ± 0.001 0.87 ± 0.04 0.91 + 0.05 D253 1.24 + 0.04 0.047 + 0.001 0.78 ± 0.04 0.94 ± 0.05 Appendix 3 226 Residue Ti (sec) T 2 [sec) NOE s2 Y254 K255 K256 V257 1 25 + 0.04 0.049 + 0.001 0.88 ± 0.04 0.91 + 0.04 V258 Q259 1 22 + 0.04 0.048 ± 0.001 0.83 ± 0.04 0.93 ± 0.05 A260 1 23 ± 0.04 0.048 ± 0.001 0.84 ± 0.04 0.93 ± 0.04 C261 1 25 ± 0.04 0.055 ± 0.002 0.84 + 0.04 0.85 ± 0.04 M262 Q263 1 24 + 0.04 0.049 ± 0.001 0.86 + 0.04 0.92 + 0.04 V264 1 29 ± 0.04 0.053 + 0.002 0.82 + 0.04 0.86 + 0.05 T265 1 19 + 0.04 0.051 ± 0.001 0.80 + 0.04 0.91 + 0.05 R266 C267 1 25 + 0.04 0.046 + 0.001 0.82 ± 0.04 0.94 + 0.05 Q268 1 30 + 0.05 0.046 ± 0.001 0.85 + 0.04 0.92 ± 0.05 G269 1 24 ± 0.04 0.048 + 0.001 0.88 ± 0.04 0.92 + 0.04 V270 1 28 ± 0.04 0.048 0.001 0.82 + 0.04 0.91 + 0.05 T271 1 39 + 0.05 0.052 + 0.002 0.86 ± 0.04 0.84 + 0.04 V272 1 26 + 0.04 0.048 ± 0.001 0.77 ± 0.04 0.91 + 0.05 W273 1 10 ± 0.04 0.046 + 0.001 0.89 + 0.04 0.99 ± 0.03 W273-lndole 1 51 + 0.06 . 0.058 ± 0.003 0.81 + 0.04 0.88 + 0.01 G274 1 23 + 0.04 0.051 + 0.001 0.88 ± 0.04 0.90 ± 0.05 I275 1 33 + 0.05 0.049 + 0.001 0.78 ± 0.04 0.89 ± 0.04 T276 D277 1 34 ± 0.05 0.043 ± 0.001 0.88 + 0.04 0.94 ± 0.05 K278 1 20 + 0.04 0.047 + 0.001 0.73 ± 0.04 0.93 + 0.04 Y279 1 33 + 0.05 0.047 + 0.001 0.75 ± 0.04 0.90 + 0.05 S280 W281 W281-Indole 1 52 + 0.30 0.059 + 0.006 0.66 + 0.03 0.86 ± 0.04 V282 1 24 ± 0.04 0.046 ± 0.001 , 0.84 ± 0.04 0.95 + 0.04 P283 D284 1 .38 ± 0.05 0.055 + 0.002 0.75 ± 0.04 0.82 + 0.05 V285 F286 1 .35 ± 0.05 0.054 + 0.002 0.72 + 0.04 0.82 + 0.05 P287 G288 1 .37 ± 0.05 0.055 + 0.002 0.81 ± 0.04 0.82 ± 0.04 E289 1 35 + 0.05 0.044 ± 0:001 0.82 + 0.04 0.93 + 0.05 G290 1 30 + 0.05 0.052 + 0.001 0.83 ± 0.04 0.86 + 0.05 A291 1 .22 ± 0.04 0.054 0.002 0.80 + 0.04 0.86 ± 0.05 A292 1 .29 + 0.05 0.044 ± 0.001 0.86 + 0.04 0.96 ± 0.04 L293 1 .24 + 0.04 0.049 ± 0.001 0.87 + 0.04 0.92 ± 0.04 V294 W295 W295-lndole 1 .53 + 0.06 0.056 + 0.002 0.80 ± 0.04 0.88 + 0.01 D296 1 .35 + 0.05 0.043 + 0.001 0.87 + 0.04 0.95 ± 0.04 A297 S298 1 .28 + 0.04 0.050 ± 0.001 0.80 + 0.04 0.89 ± 0.05 Y299 1 .20 ± 0.04 0.044 ± 0.001 0.87 + 0.04 0.97 ± 0.04 A300 ' 1 .23 ± 0.04 0.046 + 0.001 0.83 ± 0.04 0.96 + 0.04 K301 1 .34 ± 0.05 0.050 + 0.001 0.82 + 0.04 0.86 + 0.04 K302 1 .38 ± 0.05 0.047 + 0.001 0.72 ± 0.04 0.88 + 0.05 P303 Appendix 3 227 Residue ^^  (sec) T 2 (sec) NOE A304 Y305 A306 1.27 ± 0.04 0.050 + 0.001 0.83 ± 0.04 0.90 + 0.05 A307 1.22 + 0.04 0.051 ± 0.001 0.72 + 0.04 0.89 ± 0.05 V308 M309 1.27 + 0.04 0.048 + 0.001 0.74 + 0.04 0.91 ± 0.04 E310 1.21 + 0.04 0.047 + 0.001 0.88 ± 0.04 0.95 ± 0.04 A311 1.21 ± 0.04 0.053 + 0.002 0.85 ± 0.04 0.88 ± 0.05 F312 1.26 ± 0.04 0.049 ± 0.001 0.76 ± 0.04 0.90 ± 0.05 G313 1.17 + 0.04 0.059 ± 0.002 0.77 + 0.04 0.91 ± 0.07 A314 S315 Appendix 4 228 Append ix 4 N M R R e l a x a t i o n P a r a m e t e r s f o r 2 F C b - C e x C D 4 Residue Ti(sec) T 2(sec) NOE s2 A1 T2 T3 1.33 ± 0.05 0.055 + 0.002 0.71 + 0.04 0.82 + 0.05 • L4 K5 1.41 ± 0.05 0.050 ± 0.002 0.83 + 0.04 0.84 ± 0.04 E6 1.29 ± 0.04 0.044 ± 0.001 0.72 ± 0.04 0.94 + 0.04 A7 1.27 ± 0.04 0.046 ± 0.001 0.89 ± 0.04 0.93 + 0.05 A8 1.28 ± 0.04 0.050 ± 0.001 0.87 ± 0.04 0.89 ± 0.05 D9 G10 1.25 ± 0.04 0.048 + 0.001 0.87 ± 0.04 0.91 + 0.05 A11 G12 1.32 + 0.05 0.052 ± 0.002 0.86 ± 0.04 0.85 + 0.06 R13 1.40 ± 0.05 0.044 + 0.001 0.81 + 0.04 0.91 ± 0.05 , D14 1.34 + 0.05 0.043 + 0.001 0.77 + 0.04 0.94 + 0.04 F15 1.34 + 0.05 0.053 ± 0.002 0.82 ± 0.04 0.85 + 0.05 G16 F17 A18 L19 D20 P21 N22 1.22 + 0.04 0.045 + 0.001 0.78 ± 0.04 0.95 ± 0.05 R23 1.31 + 0.05 0.042 ± 0.001 0.71 + 0.04 0.95 + 0.03 L24 S25 .1.25 ± 0.04 0.045 ± 0.001 0.84 + 0.04 0.95 + 0.04 E26 1.34 + 0.05 0.046 ± 0.001 0.85 + 0.04 0.91 ± 0.05 A27 1.30 + 0.04 0.046 ± 0.001 0.85 ± 0.04 0.92 + 0.05 Q28 Y29 1.35 + 0.05 0.048 ± 0.001 0.82 + 0.04 0.89 ± 0.05 K30 1.26 ± 0.04 0.042 ± 0.001 0.83 ± 0.04 0.98 ± 0.03 A31 1.25 + 0.04 0.047 ± 0.001 0.82 ± 0.04 0.93 + 0.05 I32 1.27 ± 0.04 0.044 + 0.001 0.81 ± 0.04 0.96 ± 0.04 A33 1.14 ± 0.04 0.040 ± 0.001 0.73 + 0.04 0.98 ± 0.03 D34 1.27 + 0.04 0.047 ± 0.001 0.76 ± 0.04 0.92 ± 0.04 S35 E36 1.24 + 0.04 0.046 ± 0.001 0.82 ± 0.04 0.94 ± 0.05 F37 N38 L39 1.36 + 0.05 0.045 + 0.001 0.87 + 0.04 0.91 + 0.05 V40 V41 A42 1.25 ± 0.04 0.046 + 0.001 0.85 ± 0.04 0.93 0.04 E43 1.28 ± 0.04 0.049 ± 0.001 0.79 ± 0.04 , 0.89 + 0.05 N44 A45 1.13 + 0.04 0.036 + 0.001 0.80 + 0.04 1.00 ± 0.03 4 List of the relaxation parameters determined for 2FCb-CexCD at pH 6.5 and 30 °C. Appendix 4 229 Residue (sec) T 2(sec) NOE s 2 M46 1.39 + 0.05 0.050 ± 0.002 0.89 ± 0.04 0.85 + 0.05 K47 W48 1.36 + 0.05 0.048 + 0.001 0.81 ± 0.04 0.88 + 0.05 W48-lndole 1.61 + 0.04 0.054 + 0.001 0.81 + 0.04 0.88 + 0.03 D49 1.26 + 0.04 0.044 ± 0.001 0.89 0.04 0.96 + 0.04 A50 1.46 + 0.05 0.051 + 0.002 0.81 ± 0.04 0.83 ± 0.05 T51 1.40 ± 0.05 0.051 ± 0.002 0.85 + 0.04 0.84 ± 0.05 E52 P53 S54 1.33 ± 0.05 0.047 ± 0.001 0.87 ± 0.04 0.90 + 0.05 Q55 1.34 ± 0.05 0.051 ± 0.002 0.89 ± 0.04 0.86 + 0.05 N56 1.34 ± 0.05 0.050 ± 0.002 0.77 ± 0.04 0.86 + 0.05 S57 1.35 + 0.05 0.047 ± 0.001 0.84 + 0.04 0.90 + 0.05 F58 1.41 " ± 0.05 0.054 ± 0.002 0.77 + 0.04 0.81 + 0.05 S59 1.37 + 0.05 0.047 ± 0.001 0.89 + 0.04 0.89 ± 0.05 F60 1.49 ± 0.06 0.049 ± 0.001 0.76 + 0.04 0.84 + 0.05 G61 1.36 + 0.05 0.046 ± 0.001 0.83 ± 0.04 0.90 + 0.05 A62 1.27 ± 0.04 0.043 ± 0.001 0.79 ± 0.04 0.96 + 0.04 G63 1.40 ± 0.05 0.046 ± 0.001 0.83 + 0.04 0.89 ± 0.06 D64 1.32 ± 0.05 0.046 ± 0.001 0.81 ± 0.04 0.91 + 0.05 R65 1.29 + 0.05 0.048 ± 0.001 0.78 + 0.04 0.89 ± 0.05 V66 1.43 ± 0.05 0.043 ± 0.001 0.86 + 0.04 0.92 ± 0.05 A67 1.40 + 0.05 0.049 ± 0.001 0.88 ± 0.04 0.85 + 0.05 S68 1.31 ± 0.05 0.043 ± 0.001 0.82 + 0.04 0.95 + 0.05 Y69 1.35 ± 0.05 0.045 ± 0.001 0.85 + 0.04 0.91 ± 0.05 A70 1.30 ± 0.05 0.041 ± 0.001 0.78 ± 0.04 0.99 + 0.04 A71 1.33 ± 0.05 0.044 ± 0.001 0.89 + 0.04 0.93 ± 0.05 D72 1.34 ± 0.05 0.044 ± 0.001 0.85 + 0.04 0.93 + 0.04 T73 1.39 + 0.05 0.045 ± 0.001 0.84 ± 0.04 0.91 ± 0.05 G74 1.40 ± 0.05 0.045 ± 0.001 0.74 + 0.04 0.89 + 0.05 K75 1.37 + 0.05 0.047 ± 0.001 0.85 ± 0.04 0.89 ± 0.05 E76 1.42 ± 0.05 0.044 + 0.001 0.82 + 0.04 ' 0.91 ± 0.05 L77 1.32 ± 0.05 0.047 ± 0.001 0.81 + 0.04 0.91 + 0.05 Y78 G79 H80 1.24 ± 0.04 0.045 ± 0.001 0.88 + 0.04 0.95 + 0.05 -T81 1.39 ± 0.05 0.047 ± 0.001 0.78 + 0.04 0.89 + 0.05 L82 1.40 + 0.05 0.044 ± 0.001 0.87 + 0.04 0.92 + 0.05 V83 1.46 + 0.05 0.048 ± 0.001 0.89 + 0.04 0.86 + 0.05 W84 1.35 + 0.05 0.048 + 0.001 0.89 + 0.04 0.88 ± 0.05 W84-lndole 1.50 ± 0.05 0.053 ± 0.001 0.84 + 0.04 0^ 92 + 0.03 H85 S86 Q87 L88 P89 D90 1.30 + 0.04 0.046 ± 0.001 0.86 + 0.04 0.92 + 0.05 W91 1.22 + 0.04 0.045 ± 0.001 0.85 ± 0.04 0.95 ± 0.04 W91 -Indole 1.57 0.01 0.050 0.001 0.79 0.04 0.92 0.03 A92 1.37 + 0.05 0.047 + 0.001 0.88 ± 0.04 0.88 + 0.05 K93 1.31 + 0.05 0.046 ± 0.001 0.84 ± 0.04 0.91 + 0.05 N94 1.25 ± 0.04 0.050 ± 0.001 0.74 ± 0.04 0.89 ± 0.05 L95 1.49 + 0.06 0.046 ± 0.001 0.82 + 0.04 0.87 + 0.05 Appendix 4 230 Residue Ti(sec) T 2(sec) NOE s2 N96 1.34 + 0.05 0.045 ± 0.001 0.76 + 0.04 0.92 + 0.05 G97 1.15 + 0.04 0.055 ± 0.002 0.70 + 0.03 0.89 ± 0.04 S98 A99 1.17 ± 0.04 0.048 + 0.001 0.88 + 0.04 0.94 + 0.05 F100 1.34 + 0.05 0.045 + 0.001 0.88 + 0.04 0.92 ± 0.05 E101 1.37 ± 0.05 0.042 ± 0.001 0.79 ± 0.04 0.94 ± 0.05 S102 1.39 ± 0.05 0.045 + 0.001 0.88 + 0.04 0.90 + 0.05 A103 1.26 ± 0.04 0.045 + 0.001 0.78 + 0.04 0.94 + 0.05 M104 1.34 ± 0.05 0.045 ± 0.001 0.73 + 0.04 0.90 + 0.05 V105 1.31 + 0.05 0.041 + 0.001 0.89 + 0.04 0.98 ± 0.04 N106 1.29 ± 0.05 0.042 + 0.001 0.87 ± 0.04 0.97 + 0.04 H107 1.27 + 0.04 0.043 ± 0.001 0.89 + 0.04 0.96 + 0.04 V108 T109 K110 1.34 + 0.05 0.052 + 0.002 0.69 ± 0.03 0.83 ± 0.05 V111 A112 D113 H114 F115 1.36 + 0.05 0.050 ± 0.001 0.86 ± 0.04 0.86 + 0.05 E116 ± ± + ± G117 1.30 ± 0.05 0.048 ± 0.001 0.81 + 0.04 0.91 ± 0.05 K118 1.47 ± 0.06 0.044 ± 0.001 0.86 + 0.04 0.90 + 0.05 V119 1.57 ± 0.06 0.048 + 0.001 0.76 + 0.04 0.82 + 0.04 A120 1.34 + 0.05 0.047 + 0.001 0.88 + 0.04 0.89 + 0.05 S121 1.36 + 0.05 0.050 + 0.002 0.84 + 0.04 0.86 ± 0.05 W122 W122-lndole 1.47 ± 0.06 0.056 + 0.002 0.80 + 0.04 0.91 + 0.03 D123 V124 1.24 ± 0.04 0.039 + 0.001 0.78 + 0.04 1.00 ± 0.03 V125 N126 E127 1.33 ± 0.05 0.039 ± 0.001 0.75 + 0.04 1.00 + 0.03 A128 F129 A130 1.27 ± 0.04 0.049 + 0.001 0.86 + 0.04 0.89 + 0.05 D131 1.35 ± 0.05 0.048 ± 0.001 0.84 ± 0.04 0.88 + 0.05 G132 G133 1.30 ± 0.05 0.050 + 0.001 0.83 + 0.04 0.88 ± 0.05 G134 1.00 + 0.03 0.054 + 0.002 0.80 ± 0.04 0.69 ± 0.10 R135 R136 1.29 ± 0.04 0.051 ± 0.002 0.87 ± 0.04 0.87 ± 0.05 Q137 1.33 ± 0.05 0.050 ± 0.002 0.71 + 0.04 0.85 + 0.05 D138 S139 A140 1.18 + 0.04 0.040 ± 0.001 0.82 + 0.04 1.00 + 0.03 F141 Q142 1.34 ± 0.05 0.041 ± 0.001 0.83 ± 0.04 0.96 ± 0.04 Q143 K144 L145 G146 N147 Appendix 4 231 Residue Ti(sec) T 2(sec) NOE s2 G148 Y149 1150 E151 T152 1.28 ± 0.04 0.047 ± 0.001 0.83 ± 0.04 0.92 + 0.05 A153 F154 R155 A156 1.25 ± 0.04 0.046 ± 0.001 0.85 ± 0.04 0.94 ± 0.05 A157 1.39 ± 0.05 0.050 + 0.002 0.73 ± 0.04 0.85 + 0.04 R158 1.40 ± 0.05 0.046 ± 0.001 0.78 ± 0.04 0.89 + 0.05 A159 A160 1.34 ± 0.05 0.048 ± 0.001 0.81 + 0.04 0.89 ± 0.05 D161 1.39 + 0.05 0.049 + 0.001 0.83 + 0.04 0.87 + 0.05 P162 T163 1.30 + 0.05 0.052 ± 0.002 0.78 + 0.04 0.86 ± 0.05 • A164 1.35 ± 0.05 0.049 ± 0.001 0.73 + 0.04 0.86 + 0.05 K165 1.41 ± 0.05 0.048 ± 0.001 0.84 ± 0.04 0.87 ± 0.05 L166 C167 1.41 ± 0.05 0.043 + 0.001 0.87 ± 0.04 0.92 + 0.05 1168 N169 D170 Y171 1.35 ± 0.05 0.041 ± 0.001 0.85 ± 0.04 0.96 ± 0.04 N172 1.33 0.05 0.045 ± 0.001 0.88 ± 0.04 0.92 + 0.04 V173 1.25 + 0.04 0.046 ± 0.001 0.87 + 0.04 0.94 + 0.04 E174 1.31 + 0.05 0.050 ± 0.001 0.83 •+ 0.04 0.88 0.05 G175 1.49 ± 0.06 0.050 + 0.001 0.83 + 0.04 0.83 + 0.05 1176 N177 1.39 ± 0.05 0.051 ± 0.002 0.80 ± 0.04 0.84 ± 0.05 A178 K179 1.34 + 0.05 0.050 ± 0.002 0.80 + 0.04 0.86 ± 0.05 S180 1.31 + 0.05 0.041 + 0.001 0.83 + 0.04 0.98 + 0.04 N181 1.24 + 0.04 0.046 ± 0.001 0.85 + 0.04 0.95 ± 0.04 S182 L183 Y184 D185 1.20 + 0.04 0.042 + 0.001 0.80 + 0.04 1.00 + 0.03 L186 1.35 ± 0.05 0.048 ± 0.001 0.85 + 0.04 0.89 ± 0.05 V187 1.25 + 0.04 0.051 ± 0.002 0.87 + 0.04 0.88 ± 0.05 K188 1.32 ± 0.05 0.045 + 0.001 0.77 0.04 0.93 + 0.05 D189 F190 1.45 ± 0.05 0.044 ± 0.001 0.87 + 0.04 0.90 + 0.05 K191 1.30 ± 0.05 0.044 ± 0.001 0.81 + 0.04 0.95 + 0.04 A192 1.28 ± 0.04 0.048 + 0.001 0.84 0.04 0.91 ± 0.05 R193 1.37 + 0.05 0.047 ± 0.001 0.86 + 0.04 0.89 + 0.04 G194 1.37 ± 0.05 0.050 ± 0.001 0.77 ± 0.04 0.85 + 0.05 V195 1.26 + 0.04 0.048 + 0.001 0.83 + 0.04 0.92 ± 0.05 P196 L197 D198 1.28 ± 0.05 0.047 + 0.001 0.86 + 0.04 0.92 + 0.05 C199 V200 Appendix 4 232 Residue Ti(sec) T 2(sec) NOE s2 G201 1.30 ± 0.05 0.041 + 0.001 0.84 + 0.04 0.98 + 0.04 F202 _Q203 S204 1.34 + 0.05 0.045 + 0.001 0.84 + 0.04 0.92 + 0.05 H205 L206 . 1.36 ± 0.05 0.052 ± 0.002 0.80 ± 0.04 0.84 + 0.05 I207 1.35 ± 0.05 0.051 + 0.002 0.81 ± 0.04 0.86 ± 0.05 V208 1.26 + 0.04 0.052 ± 0.002 0.88 + 0.04 0.87 + 0.05 G209 1.33 + 0.05 0.050 + 0.002 0.86 ± 0.04 0.87 ± 0.05 Q210 1.30 ± 0.05 0.047 + 0.001 0.81 + 0.04 0.91 + 0.05 V211 1.44 ± 0.05 0.047 + 0.001 0.77 ± 0.04 0.86 + 0.05 P212 G213 1.43 + 0.05 0.047 + 0.001 0.85 + 0:04 0.88 + 0.05 D214 1.23 ± 0.04 0.043 ± 0.001 0.82 + 0.04 0.99 ± 0.04 F215 1.34 + 0.05 0.042 + 0.001 0.82 ± 0.04 0.96 + 0.04 R216 1.28 + 0.04 0.043 ± 0.001 0.83 + 0.04 0.97 + 0.04 Q217 1.31 + 0.05 0.046 + 0.001 0.83 ± 0.04 0.92 + 0.05 N218 1.35 + 0.05 0.042 + 0.001 0.88 ± 0.04 0.95 + 0.04 L219 Q220 1.38 ± 0.05 0.041 + 0.001 0.80 ± 0.04 0.96 ± 0.04 R221 1.34 ± 0.05 0.043 ± 0.001 0.82 ± 0.04 0.95 ± 0.04 F222 A223 1.42 ± 0.05 0.049 + 0.002 0.70 ± 0.04 0.84 + 0.05 D224 L225 1.26 + 0.04 0.043 + 0.001 0.78 + 0.04 0.97 + 0.04 G226 1.39 ± 0.05 0.044 ± 0.001 0.86 + 0.04 0.91 + 0.05 V227 1.39 ± 0.05 0.054 + 0.002 0.80 ± 0.04 0.81 + 0.05 D228 V229 1.30 ± 0.05 0.045 ± 0.001 0.85 ± 0.04 0.93 + 0.05 R230 1231 1.19 + 0.04 0.039 + 0.001 0.76 ± 0.04 1.00 + 0.03 T232 E233 L234 1.21 + 0.04 0.047 ± 0.001 0.77 ± 0.04 0.94 ± 0.04 D235 I236 R237 M238 1.23 + 0.04 0.043 ± 0.001 0.84 ± 0.04 0.99 ± 0.04 R239 1.33 ± 0.05 0.048 + 0.001 0.81 ± 0.04 0.88 ± 0.05 T240 P241 S242 D243 A244 T245 1.21 ± 0.04 0.055 + 0.002 0.71 ± 0.04 0.85 ± 0.05 K246 1.33 ± 0.05 0.047 ± 0.001 0.88 ± 0.04 0.91 • ± 0.05 L247 1.32 + 0.05 0.043 + 0.001 0.88 + 0.04 0.96 ± 0.04 A248 1.23 ± 0.04 0.047 ± 0.001 0.86 ± 0.04 0.93 ± 0.05 T249 Q250 A251 1.29 + 0.05 0.048 + 0.001 0.84 + 0.04 • 0.90 ± 0.05 A252 1.28 + 0.04 0.044 ± 0.001 0.85 ± 0.04 0.95 ± 0.05 D253 Appendix 4 233 Residue (sec) T 2 [sec) NOE S2 Y254 K255 K256 > V257 1.25 + 0.04 0.047 + 0.001 0.80 + 0.04 0.93 ± 0.05 V258 1.38 ± 0.05 0.046 ± 0.001 0.71 ± 0.04 0.88 ± 0.04 Q259 1.35 + 0.05 0.045 ± 0.001 0.86 + 0.04 0.92 ± 0.05 A260 1.30 + 0.05 0.045 + 0.001 0.86 + 0.04 0.94 ± 0.04 C261 1.46 + 0.05 0.047 ± 0.001 0.84 ± 0.04 0.87 + 0.04 M262 1.13 + 0.04 0.052 + 0.002 . 0.72 + 0.04 0.89 + 0.05 Q263 1.29 + 0.04 0.046 + 0.001 0.86 + 0.04 0.93 ± 0.05 V264 1.30 + 0.05 0.051 + 0.002 0.82 + 0.04 0.87 + 0.05 T265 1.18 + 0.04 0.049 ± 0.001 0.77 ± 0.04 0.92 + 0.05 R266 1.25 ± 0.04 0.047 ±0.001 0.78 ± 0.04 0.93 ± 0.05 C267 1.31 ± 0.05 0.044 ±0.001 0.86 ± 0.04 0.94 + 0.04 Q268 G269 1.30 ± 0.05 0.046 ± 0.001 0.88 + 0.04 0.92 + 0.05 V270 T271 1.38 + 0.05 0.052 + 0.002 0.75 ± 0.04 0.84 ± 0.04 V272 W273 1.37 ± 0.05 0.046 ± 0.001 0.88 + 0.04 0.90 ± 0.05 W273-lndole 1.64 ± 0.17 0.049 ± 0.003 0.85 ± 0.04 0.92 ± 0.04 G274 I275 1.49 ± 0.06 0.047 ± 0.001 0.79 ± 0.04 ' 0.86 ± 0.05 T276 D277 1.28 + 0.04 0.041 ± 0.001 0.86 ± 0.04 0.98 + 0.04 K278 1.22 + 0.04 0.043 + 0.001 0.82 + 0.04 0.98 + 0.04 Y279 1.33 + 0.05 0.044 + 0.001 0.82 0.04 0.94 + 0.05 S280 W281 1.24 + 0.04 0.047 + 0.001 0.88 + 0.04 0.94 + 0.04 W281-Indole 1.50 ± 0.09 0.059 ± 0.001 0.84 ± 0.04 0.87 ± 0.03 V282 P283 D284 1.37 0.05 0.049 ± 0.001 0.78 ± 0.04 0.88 ± 0.06 V285 1.46 + 0.05 0.048 ± 0.001 0.74 ± 0.04 0.85 ± 0.05 F286 1.36 + 0.05 0.047 ±. 0.001 0.81 ± 0.04 . 0.90 ± 0.04 P287 G288 1.36 + 0.05 0.052 ± .0.002 0.80 + 0.04 0.84 + 0.05 E289 1.36 + 0.05 0.043 ± 0.001 0.84 + 0.04 0.94 + 0.05 G290 A291 1.29 + 0.04 0.051 ± 0.002 0.75 ± 0.04 0.87 ± 0.05 A292 1.34 + 0.05 0.042 ± 0.001 0.88 ± 0.04 0.96 ± 0.04 L293 1.28 + 0.04 0.049 ± 0.001 0.89 ± 0.04 0.90 ± 0.05 V294 1.21 ± 0.04 0.038 ± 0.001 0.73 ± 0.04 0.98 + 0.03 W295 1.31 ± 0.05 0.039 ± 0.001 0.82 ± 0.04 1.00 ± 0.03 W295-lndole 1.54 + 0.02 0.054 ± 0.001 0.77 ± 0.04 0.90 ± 0.03 D296 A297 S298 1.36 + 0.05 0.047 ± 0.001 0.81 ± 0.04 0.90 ± 0.05 Y299 + ± + ± A300 1.22 + 0.04 0.044 ± 0.001 0.83 + 0.04 0.97 + 0.04 K301 1.39 ± 0.05 0.048 ± 0.001 0.84 + 0.04 0.87 ± 0.05 . K302 1.48 + 0.06 0.046 ± 0.001 0.80 + 0.04 0.87 + 0.05 P303 Appendix 4 234 Residue Ti(sec) T 2(sec) NOE s2 A304 Y305 1.27 ± 0.04 0.043 ± 0.001 0.83 + 0.04 0.97 + 0.04 A306 1.33 ± 0.05 0.049 ± 0.001 076 ± 0.04 0.89 ± 0.05 A307 1.27 ± 0.04 0.047 ± 0.001 0.84 ± 0.04 0.93, ± 0.04 V308 1.42 ± 0.05 0.045 ± 0.001 0.74 ± 0.04 0.89 ± 0.05 M309 E310 1.24 ± 0.04 0.042 ± 0.001 0.79 ± 0.04 0.99 + 0.03 A311 1.25 ± 0.04 0.049 ± 0.001 0.81 ± 0.04 0.90 ± 0.05 F312 G313 1.21 + 0.04 0.054 ± 0.002 0.73 ± 0.04 0.85 ± 0.05 A314 0.95 ± 0.03 0.066 + 0.002 0.70 + 0.04 0.76 + 0.05 S315 

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