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Specific interactions between carbohydrate-binding modules and cellulose McLean, Bradley William 2001

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Specific interactions between carbohydrate-binding modules and cellulose by BRADLEY WILLIAM MCLEAN M.Sc, Queen's University, 1995 B.Sc .H. , Queen's University, 1991 A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF D O C T O R O F P H I L O S O P H Y in T H E FACULTY OF GRADUATE STUDIES (DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY AND T H E BIOTECHNOLOGY LABORATORY) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA Apr i l 2001 © Bradley Wil l iam McLean, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of MK*09IQIJ*V AUQ J-*\AM»L<t6-j The University of British Columbia Vancouver, Canada Date Atou 2*>' ii Abstract Polysaccharidases and the carbohydrate-binding modules (CBMs) have evolved to mirror the diversity inherent in the crystalline and amorphous structures of cellulose and other polysaccharides abundant in nature. In this study, the binding specificities for cellulose of C B M s representing each of the three functional types of C B M s (types A , B and C) , were determined by a series of pair-wise competition binding experiments. These studies show that different binding sites of phosphoric acid-swollen cellulose (PASC) are bound by different C B M s : type A localize to crystalline regions, type B localize to the amorphous regions. The data suggest that there is heterogeneity in the amorphous regions of P A S C . The binding of the family 2a C B M from Cellulomonas fimi xylanase 10A (CBM2a) to insoluble cellulose was characterized in more detail. Competition experiments indicate that C B M 2 a binds to the crystalline regions of cellulose. It has been reported that C B M 2 a binds irreversibly to cellulose however, it is shown here that C B M 2 a bound to the cellulose surface can be displaced by C B M 2 a in solution. C B M 2 a has a number of conserved residues on the binding face, including three tryptophans that have been implicated in the binding reaction. Site-directed mutation and a Langmuir-type adsorption isotherm analysis was used to determine the individual contributions of W17, W54, W72 and a number of neighbouring residues to the overall binding affinity of C B M 2 a for cellulose. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. In an attempt to formulate a structural model of C B M 2 a bound to the surface of cellulose, observation of l 5 N -labelled C B M 2 a bound to 1 3C-enriched cellulose using solid state N M R was attempted. Given the specificity of C B M 2 a , the structure of crystalline cellulose and the dynamic nature of the binding of C B M 2 a , a model is proposed for the interaction between the polypeptide and the crystalline surface that is also likely to apply to other type A C B M s . iv Table of Contents A B S T R A C T ii T A B L E O F C O N T E N T S iv L I S T O F T A B L E S viii L I S T O F F I G U R E S ix A B B R E V I A T I O N S xi S T A T E M E N T O F P U B L I C A T I O N xii S T A T E M E N T O F C O L L A B O R A T I O N xiii A C K N O W L E D G E M E N T S xiv 1. I N T R O D U C T I O N 1 1.1 CELLULOSE l 1.2 POLYSACCHARIDE HYDROLASES AND CELLULASES 2 1.3 CARBOHYDRATE-BINDING MODULES 4 1.3.1 CBM nomenclature 5 1.3.2 Type A CBMs 6 1.3.2.1 Family 1 6 1.3.2.2 Family 2a 7 1.3.2.3 Family 3 9 1.3.2.4 Family 5 10 1.3.2.5 Family 10 11 1.3.3 Type B CBMs 13 1.3.3.1 Family 2b 13 1.3.3.2 Family 4 14 1.3.3.4 Family 17 15 1.3.4 Type C CBMs 17 1.3.4.1 Family 9 17 1.3.5 Applications of CBMs 20 l .5 GOAL OF THIS RESEARCH 20 2. M A T E R I A L S A N D M E T H O D S 22 2.1 MATERIALS AND REAGENTS 22 V 2.2 BACTERIAL STRAINS AND PLASMIDS 22 2.2.1 Escherichia coli strains and plasmids 22 2.2.2 Acetobacter xylinum strains 25 2.3 CULTURE MEDIA 23 2.3.1 E. coli growth media 23 2.3.2 A. xylinum growth media 24 2.4 DNA MANIPULATIONS 24 2.4.1 Synthesis of DNA fragment encoding CBM2a 25 2.4.2 Construction ofCBM2a mutants 27 2.5 PRODUCTION AND PURIFICATION OF CBMs 28 2.5.1 Production of CBMs used in competition isotherms 28 2.5.2 Production and purification ofCBM2a and mutants 29 2.5.2.1 M9 Media for production of l 5N-CBM2a 30 2.5.2.2 Production of l5N-labelled CB.\12a 31 2.6 PRODUCTION AND PROCESSING OF BACTERIAL CELLULOSE 31 2.6.1 Production ofl3C-enriched Cellulose 31 2.6.1.1 Production of l3C-4-enriched Cellulose 31 2.6.1.2 Production ofl,3,4,6-l3C-enriched cellulose 32 2.6.2 Processing of bacterial cellulose 33 2.7 DETERMINATION OF AFFINITIES 33 2.8 COMPETITION BINDING ISOTHERMS 34 2.8.1 Labelling of CBMs with fluorescent probes 34 2.8.2 Standard curves of fluorescence vs. polypeptide concentration 35 2.8.3 Isotherms by fluorescence 36 2.8.4 Competition isotherms 37 2.9 ADSORPTION AND SURFACE EXCHANGE OF C B M 2 A TO CELLULOSE 37 2.9.1 Surface exchange of CBM2a 37 2.9.2 Kinetics of adsorption to PASC and BMCC 38 2.9.3 Kinetics ofCBM2a exchange on BMCC 39 vi 2.10 N M R 40 2.10.1 Solid state NMR: Cross-polarization/magic angle spinning (CP/MAS) 40 2.10.2 NMR in solution 41 3. RESULTS 42 3.1 PRODUCTION O F H I S 6 - C B M 2 A 42 3.2 BINDING OF C B M 2 A TO C E L L U L O S E 43 3.3 BINDING OF C B M 2 A M U T A N T S T O B M C C 49 3.3.1 Tryptophan mutants 49 3.3.2 Other mutants 50 3.4 COMPETITION BINDING ISOTHERMS 52 3.4.1 CBMs bind to PASC and BMCC 52 3.4.2 Labelling of CBMs 53 3.4.3 Standards and Isotherms by fluorescence 54 3.4.4 CBM2a adsorption to PASC in the presence ofBSA 56 3.4.5 A model of competition binding 61 3.4.6 CBM2a in competition with CBM4-1, CBM 17 and CBM9-2 66 3.4.7 CBM2a in competition with Cel6A-CBM2a and CBM3 73 3.4.8 CBM4-1 and CBM 17 competition 78 3.5 C B M 2 A BINDING ADSORPTION A N D E X C H A N G E 81 5.5.7 Rate of Adsorption of CBM2a to PASC and BMCC 81 3.5.2 Equilibrium exchange ofCBM2a on BMCC 81 3.5.3 Rate of CBM2a exchange on BMCC 83 3.6 C B M 2 A - C E L L U L O S E INTERACTION O B S E R V E D B Y SOLID S T A T E N M R 86 3.6.1 Production and characterization of'3C-enriched bacterial cellulose 86 3.6.2 CBM2a-cellulose interaction 87 4. DISCUSSION 92 4.1 C E L L U L O S E A N D C B M S : A DIVERSITY OF STRUCTURE A N D FUNCTION 93 4.1.1 Binding specificity of CBMs for cellulose 93 vii 4.1.1.1 CBM2a binds to crystalline regions of cellulose 93 4.1.1.2 Heterogeneity of amorphous cellulose structure 97 4.1.2 CBMs: agents for targeting polysaccharidases and for saturating substrate 100 4.2 ADSORPTION OF CBM2A TO C E L L U L O S E 103 4.2.1 Irreversibility of adsorption of CBM2a 103 4.2.2 Amino acid residues involved in the binding ofCBM2a to crystalline cellulose 106 4.2.3 CBM2a-Cellulose interaction observed by solid state NMR 109 4.3 T O W A R D S A M O D E L OF C B M 2 A - C E L L U L O S E INTERACTION i l l 4.3.1 Previous models of binding Ill 4.3.2 Surface area of the exposed crystalline faces 112 4.3.3 A model for the binding ofCBM2a to crystalline cellulose 113 4.4 A G E N E R A L M O D E L FOR T H E BINDING OF T Y P E A BINDING M O D U L E S 115 R E F E R E N C E S 117 V l l l List of Tables T A B L E 2.1 OLIGONUCLEOTIDES USED FOR THE CONSTRUCTION OF THE SYNTHETIC H I S 6 - C B M 2 A GENE FRAGMENT 25 T A B L E 2.2 OLIGONUCLEOTIDES USED FOR THE CONSTRUCTION OF H I S 6 - C B M 2 A MUTANTS 28 T A B L E 2.3 CBMs USED IN COMPETITION ISOTHERMS ON PASC 29 T A B L E 3.1 BINDING AFFINITY AND CAPACITY FOR THE ADSORPTION OF C B M 2 A TO INSOLUBLE CELLULOSE. 44 T A B L E 3.2 BINDING AFFINITY OF C B M 2 A TRYPTOPHAN VARIANTS FOR B M C C 50 T A B L E 3.3 BINDING AFFINITY OF C B M 2 A VARIANTS FOR B M C C 50 T A B L E 3.4 C B M BINDING AFFINITY AND CAPACITY FOR B M C C AND PASC 53 T A B L E 3.5 CBMs, LABELS AND NUMBER OF POTENTIAL LABELLING SITES FOR CBMs USED IN COMPETITION BINDING EXPERIMENTS 54 T A B L E 3.6 BINDING OF C B M 2 A TO P A S C : UV ABSORBANCE, FLUORESCENCE AND B S A COMPETITION 56 T A B L E 3.7 CBM2A-0G IN COMPETITION WITH C B M 2 A - A X ON B M C C 63 T A B L E 3.8 PASC COMPETITION BINDING SUMMARY I: CBM2A, CBM4-1, C B M 17 AND CBM9-2 68 T A B L E 3.9 COMPETITION BINDING SUMMARY II: C B M 2 A , C E L 6 A - C B M 2 A AND CBM3 74 T A B L E 3.10 COMPETITION BINDING SUMMARY III: CBM17 A N D C B M 4 - 1 78 T A B L E 3.11 EXCHANGE AT EQUILIBRIUM OF C B M 2 A BOUND TO B M C C 82 ix List of Figures F I G U R E 1.1 M O D E L OF A PORTION OF T H E P L A N T C E L L W A L L F R O M GRASSES 2 F I G U R E 1.2 T H E C E L L U L A S E S Y S T E M OF C. FIMI 3 F I G U R E 1.3 A L I G N M E N T OF F A M I L Y 2A C B M S F R O M C. FIMI 9 F I G U R E 1.4 STRUCTURES OF T Y P E A CBMs 12 F I G U R E 1.5 STRUCTURES OF T Y P E B C B M S 16 F I G U R E 1.6 STRUCTURES OF T Y P E C C B M S 19 F I G U R E 2 . 1 RESTRICTION M A P OF T H E C B M 2 A EXPRESSION VECTOR P T U G K - H 6 - I E G R - C B M 2 A 23 F I G U R E 2.2 S C H E M A T I C FOR T H E TWO-STEP CONSTRUCTION OF T H E H I S 6 - C B M 2 A F R A G M E N T 26 F I G U R E 3.1 PURIFICATION OF H I S 6 - C B M 2 A F R O M C O N C E N T R A T E D E. COU C U L T U R E S U P E R N A T A N T 43 F I G U R E 3.2 CP/MAS SPECTRA OF INSOLUBLE C E L L U L O S E S 47 F I G U R E 3.3 B I N D I N G OF C B M 2 A TO T H E INSOLUBLE C E L L U L O S E S P A S C , B M C C A N D A V I C E L 48 F I G U R E 3.4 Two VIEWS OF C B M 2 A HIGHLIGHTING T H E M U T A T E D RESIDUES 51 F I G U R E 3.5 EMISSION SPECTRA OF C B M 2 A L A B E L L E D WITH O R E G O N G R E E N OR A M C A - X 57 F I G U R E 3.6 S T A N D A R D C U R V E S OF F L U O R E S C E N C E V S . PROTEIN CONCENTRATION : PASC EXPERIMENTS . . . . 58 F I G U R E 3.7 S T A N D A R D C U R V E S OF FLUORESCENCE VS. PROTEIN CONCENTRATION : B M C C 59 F I G U R E 3.8 B I N D I N G OF C B M 2 A - O G TO PASC: U V , FLUORESCENCE A N D BSA 60 F I G U R E 3.9 B I N D I N G OF C B M 2 A - O G TO B M C C IN T H E PRESENCE OF C B M 2 A - A X (1) 64 F I G U R E 3 .10 B I N D I N G OF C B M 2 A - O G TO B M C C IN T H E PRESENCE OF C B M 2 A - A X (2) 65 F I G U R E 3 .11 B I N D I N G OF C B M 2 A - O G TO PASC IN T H E PRESENCE OF CBM4-1 69 F I G U R E 3 .12 B I N D I N G OF CBM4-1-AX TO PASC IN T H E PRESENCE OF C B M 2 A - O G 70 F I G U R E 3 .13 B I N D I N G OF C B M 1 7 - O G TO PASC IN T H E PRESENCE OF C B M 2 A 71 F I G U R E 3 .14 B I N D I N G OF C B M 2 A - O G IN T H E PRESENCE OF CBM9-2 72 F I G U R E 3 .15 B I N D I N G OF C B M 2 A - O G TO PASC IN T H E PRESENCE OF CBM3 75 F I G U R E 3 .16 B I N D I N G OF CBM2A-0G TO PASC IN T H E PRESENCE OF C E L 6 A - C B M 2 A - A X 76 F I G U R E 3 .17 B I N D I N G OF C B M 2 A - O G TO B M C C IN T H E PRESENCE OF C E L 6 A - C B M 2 A - A X 77 F I G U R E 3 .18 B I N D I N G CBM17-0G TO PASC IN T H E PRESENCE OF CBM4-1-AX 79 F I G U R E 3 .19 B I N D I N G 0FCBM4-1-AX TO PASC IN T H E PRESENCE OF CBM17-0G 80 F I G U R E 3 .20 ADSORPTION KINETICS OF C B M 2 A BINDING TO PASC A N D B M C C 84 F I G U R E 3.21 S U R F A C E E X C H A N G E O F C B M 2 A AT EQUILIBRIUM O N B M C C 85 F I G U R E 3.22 KINETICS OF C B M 2 A S U R F A C E E X C H A N G E ON B M C C 85 F I G U R E 3.23 CP/MAS SPECTRUM OF B A C T E R I A L C E L L U L O S E F R O M G L U C O S E - l 3 C - 4 E N R I C H M E N T 89 F I G U R E 3.24 CP/MAS SPECTRA OF B M C C A N D 1 3 C - E N R I C H E D C E L L U L O S E F R O M l , 3 - 1 3 C - G L Y C E R O L SYNTHESIS 90 F I G U R E 3 .25 L 5 N CP/MAS SPECTRUM OF 1 5 N - C B M 2 A B O U N D TO INSOLUBLE C E L L U L O S E A N D HSQC OF 1 5 N -CBM2A IN SOLUTION 91 F I G U R E 4.1 CBMs B O U N D TO NATIVE C E L L U L O S E 97 F I G U R E 4.2 D E T A I L S OF T H E BINDING SITES OF CBM4-1 A N D C B M 17 99 X F I G U R E 4.3 A M O D E L OF C B M 2 A SURFACE E X C H A N G E 104 F I G U R E 4.4 T H R E E VIEWS E A C H OF TWO POSSIBLE A R R A N G E M E N T S OF C B M 2 A B O U N D TO T H E C R Y S T A L L I N E S U R F A C E OF B M C C 116 xi Abbreviations aa amino acid absorbance at wavelength "n" A M C A - X or A X 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid, succinimidyl ester B M C C bacterial micro-crystalline cellulose bp base pair, base pairs BSA bovine serum albumin CBD cellulose-binding domain C B M carbohydrate-binding module CP/MAS cross-polarization/magic-angle spinning Da Dalton DMSO dimethylsufoxide DNA deoxyribonucleic acid E H E C ethylhydroxyethylcellulose d H 2 0 distilled water dNTP deoxyribonucleoside triphosphate F W - C B M 2 a 5-fluoro-L-tryptophan-labelled C B M 2 a H&S Hestrin and Schramm medium H E C hydroxyethylcellulose H P L C high pressure liquid chromatography HSQC heteronuclear single quantum correlation K a association affinity constant kDa kiloDalton M W molecular weight NBS N-bromosuccinimide NMR nuclear magnetic resonance O G Oregon Green® 514 carboxylic acid, succinimidyl ester o/n overnight PASC phosphoric acid-swollen cellulose PCR polymerase chain reaction rpm revolutions per minute RT room temperature SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis T M S tetramethylsilane T Y P tryptone, yeast extract, phosphate medium U unit, units UV ultraviolet V volume w weight xi i Statement of Publication A portion of this thesis presents data and conclusions previously issued in the following publication: M c L e a n , B . W . , M . R . Bray , A . B . Boraston, N . R . Gi lkes , C . A . Haynes, and D . G . K i l b u r n . 2000. Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues. Protein Eng. 13: 801-809. As indicated by the order of authorship, the majority of the work presented in this publication, the competition binding experiments, construction of the eleven of the fourteen C B M 2 a mutants, and all of the binding analysis and was done by the thesis author, B . W . McLean. The synthetic C B M 2 a gene fragment was designed by R. Graham and C . Rodriguez. M . R. Bray constructed three of the fourteen C B M 2 a mutants. The modified Langmuir-type binding model presented was developed collaboratively with A . B . Boraston. N . R. Gilkes provided details and impetus for the cellulose surface area calculations. N . R. Gilkes , C. A . Haynes and D . G . Kilburn all acted in a supervisory role. D . G . Kilburn, Senior Author X l l l Statement of Collaboration The solid state N M R experimental data presented here is the product of an ongoing collaboration between the laboratories of Drs. R. A . J. Warren, Department of Microbiology and Immunology, D . G . Kilburn, Department of Microbiology and Immunology and the Biotechnology Laboratory, and Dr. Col in A . Fyfe, Department of Chemistry, U B C . Dr. C. A . Fyfe and Darren Brouwer, a Ph.D. candidate supervised by Dr. Fyfe measured all of the solid state N M R spectra presented here. A l l of the samples measured by N M R were prepared by B . W . McLean. It has been agreed by all collaborators that these results are appropriate to be included in this thesis. xiv Acknowledgements If it takes a village to raise a child, then it takes a bustling metropolis to raise a Ph.D. candidate; I wi l l attempt to express my gratitude to and acknowledge some of individuals involved in the life of this particular Ph.D. candidate. First of all, I must thank Tony Warren and Doug Kilburn, my supervisors, for providing guidance, wisdom, and humour; you set an example of excellent leadership and mentorship. Thanks are also extended to the other members of my supervisory committee, Chip Haynes and Colin Fyfe, for sharing their insights, expertise and for their enthusiastic guidance. Together, my committee leaps other supervisory committees in a single bound! I also must thank Peter Tomme, and Nei l Gilkes for their peerless expertise and their eagerness to share it. Thanks are extended to Dr. Colin Fyfe and Darren Brouwer for their enthusiastic collaboration resulting in the solid state N M R work presented here. The collection of the 1 5 N -C B M 2 a H S Q C spectrum by Cameron Mackereth and Dr. Lawrence Mcintosh is also very much appreciated. The Cellulase Lab has been a welcoming second home for the past few years. I thank all of the members of the Cellulase Lab, past and present, who help to make it a vital dynamic place to work, learn and be. A l l members of the lab have left their fingerprints on this document in some manner, I wi l l try to name at least some of them here. To all of people in room 324, past and present, thank you all for scientific inspiration for friendship (Cheers!) especially to A l Boraston, Mark Bray, Jeff Kormos, and Dominik Stoll. I am indebted to Emily Kwan and, Emily Amandorn-Akow for all of your assistance and encouragement (technical and otherwise); somewhere in this thesis is that last isotherm, thanks in no small part to your help, and to Helen Smith who kept the lab running so smoothly; your generosity and spirit is admirable. And all of the rest of you, whom I am forgetting, my thanks as well! I could not have done this without the foundation of personal support that I am lucky to have. Thank you to my parents, who have always been so very supportive of my scientific pursuits (starting at a very young age!). I am also very grateful for the support of my whole extended family and family-in law. I owe so much of this to Julie, my love and my Gibraltar, who has been a source of unfaltering support and inspiration. With you, I am always reminded that I am truly blessed and lucky. 1 1. Introduction 1.1 Cellulose Cellulose, the major structural component of plant material, is the most abundant biopolymer on earth; each year plants synthesize about 4 x 109 tonnes (21). Cellulose is also produced by certain bacteria, marine invertebrates, fungi, slime moulds and amoebae (99). Cellulose is a polymer of P-l,4-linked glucose units; in nature, the cellulose molecules are aggregated together, in parallel, in the lattice forming microfibrils. The lateral dimensions of the microfibrils range in size from 3 nm to 20 nm depending on the biological source (18). Despite its chemically simple structure, cellulose can assume a diversity of complex structures or allomorphs, with unique secondary and tertiary conformations (2). Naturally produced cellulose, cellulose I, itself is a mixture of two structural forms, I a and I p (3). Other allomorphs, such as cellulose III and the antiparallel cellulose II, arise after subjecting natural cellulose to harsh chemical treatments (2). In the plant cell wall, cellulose is intimately associated with other polysaccharides and lignin. Collectively, the other asscociated polysaccharides are called hemicellulose and may include, but are not restricted to, mannans, galactomanans, xylan, xyloglucans, pectin and some glucans (Figure 1.1). A l l of these polysaccharides are arranged in the plant cell wall in close proximity to each other, with domains of one type of polysaccharide, such as cellulose, for example, immediately adjacent to, or embedded in a matrix of another polysaccharide, such as xylan and xyloglucan, for example. The precise arrangement and composition of cellulose depends on plant species. 2 Figure 1.1 Model of a portion of the plant cell wall from grasses. This model shows the complexity and intimiate association of the component polysaccharides, such as xyloglucans and cellulose (15). The inset figure is a model of a fibril, with cellulose microfibrils associated with a matix of hemicelluloses and lignin (adapted from http://www.chemistry.vt.edu/chem-dept/helm/3434WOOD/notesl/morphology.pdf) 1.2 Polysaccharide Hydrolases and Cellulases Organisms have evolved diverse strategies for taking advantage of the rich and abundant carbon sources found in plant materials by using a complement of polysaccharide hydrolases (134). Polysaccharidase enzyme systems act in a concerted manner, often synergistically, to degrade natural substrates (26, 86, 134). A n excellent example of a well characterized polysaccharidase system is the complement of enzymes from the cellulase system of the gram-positive aerobic soil bacterium Cellulomonas fimi (Figure 1.2). 3 Cel6A I * 1//////////A Ce.9A Y/////////////////A 3 I I I I ^ i Ce.98 1 _ : i ^ r ^ Cel5A V////////A I I 2^  i «« V//////////////A I I I ^ I Ce-48A V/////////////////////X I I I ^ 1 XynlOA Y//////////A^\ Figure 1.2 The cellulase system of C.fimi. This schematic presents the major cellulases produced by C.fimi: Ce l6A (formerly CenA), Ce l9A (CenB), Cel9B (CenC), Ce l5A (CenD), Cel6B (CbhA), Cel48A (CbhB) and X y n l O (Cex). The key to domain and module structures of C. fimi polysaccharidases is provided in the figure. The C.fimi cellulases, like other polysaccharidases, are modular (36). Each enzyme is comprised of a series of distinct contiguous amino acid sequences that fold into discrete modules and are often separated by linker sequences (Figure 1.2). The modules include catalytic modules and ancillary modules, the most common of which are substrate binding modules. Cellulases themselves are an important subject of study because of their the potential application in the commercial conversion of biomass into fermentable sugars, as well as other industrial processes such as the clarification of fruit juices, and as additives to laundry detergents for the removal of small fibres, thus brightening the colours. Catalytic Module Fn3 module 4 1.3 Carbohydrate-binding modules Amongst the ancillary modules comprising cellulases, the most common type mediates binding to substrate (36, 129). The first of the substrate-binding modules characterized bound to cellulose and were known as cellulose-binding domains (CBDs). A large number of other substrate-binding modules have been discovered with binding specificity for polysaccharides other than cellulose, so these are now collectively referred to as carbohydrate-binding modules (CBMs) . Within the polysaccharidases, they can be located at either the N-or C-termini of the enzyme or internally; they can appear singly or in multiples, and may also occur in conjunction with C B M s from other families. The discrete modular structure of C B M s makes them amenable to study as functioning units isolated from the source enzyme by either limited proteolysis or genetic manipulation. C B M s are classified into at least 17 families of related amino acid sequences (24). The families themselves can be grouped into three types based on structure, function and ligand-binding specificity. Type A C B M s (Figure 1.4) bind to insoluble surfaces, type B C B M s (Figure 1.5) bind to oligosaccharides having more than three monosaccharide units and type C (Figure 1.6) bind to mono- and disaccharides (9). A major role of C B M s is to promote the hydrolysis of insoluble substrates by increasing the local concentration of the enzyme on the substrate (74, 82); the role of C B M s that bind to soluble sugars is not entirely clear. Removal of C B M s from enzymes reduces their activities on insoluble substrates; hydrolysis of soluble substrates is not affected (38, 47, 98, 128). C f C e l 6 A - C B M 2 a has been shown to disrupt the surface of fibres of ramie cotton and release small particles and thus may further increase the available surface area accessible to an organism's hydrolases (28). This effect, however, has not been demonstrated with other C B M s . Furthermore, C B M s may promote the hydrolysis of substrate (16) by targeting the 5 partner hydrolases to specific types of ligands. This diversity of C B M binding characteristics could ensure that, given a substrate that is both heterogeneous in composition and structure such as that of the plant cell wall, the maximum substrate surface area available to the organism is covered by its secreted polysaccharidases. 1.3.1 C B M n o m e n c l a t u r e As yet there is not a consensus on the naming of C B M s , with research groups tending to use an idiosyncratic approach. With the explosion in the number of C B M s characterized, and the advent of high throughput methods of protein discovery and characterization, it is imperative to have a standard system for naming them so that each one can be uniquely identified. Our group has proposed a system of C B M nomenclature (9) that is consistent with the accepted glycosyl hydrolase designation scheme (50); in both schemes, polypeptides are grouped into families of related amino acid sequences. The scheme for C B M s was refined recently. For the purposes of this work, the following convention wi l l be followed: a two letter abbreviation of the source organism (such as " C f ' for C. fimi), followed by the identifier of the parent enzyme or polypeptide designation (based on the polysaccharidase classification system (50)) then " C B M " , for "carbohydrate-binding module", the family designation and binding module number, i f more than one module of any particular family type occurs in the same enzyme or protein. That is, <two letter abreviation of source organismxglycosyl hydrolase source name>-CBM<carbohydrate-binding module f a m i l y x C B M module number if any>. For example, the family 2a binding module from the Cellulomonas fimi family 10 xylanase A , (formerly C B D C e x ) would be designated as C f X y n l 0 A - C B M 2 a . The first N -terminal binding module from the C.fimi family 9 cellulase B (formerly C B D N 1 ) is CfCel9B-C B M 4 - 1 , the second N-terminal binding module is C f C e l 9 B - C B M 4 - 2 (formerly C B D N 2 ) ; a construct containing both family 4 binding modules would be CfCe l9B-CBM4-1 .2 . Once 6 defined, these examples could be abbreviated to C B M 2 a , C B M 4 - 1 , C B M 4 - 1 and C B M 4 -1.2, respectively. 1.3.2 T y p e A C B M s This type of C B M ranges in size from 35 aa to 140 aa and includes the C B M s from families 1, 2a, 3, 5, 10 and 12. They share an affinity for insoluble cellulose, with insignificant affinity for soluble sugars or cello-oligosaccharides. Structurally, they all have a platform of aromatic residues aligned along one face of the globular polypeptide (Figure 1.4). These aromatic residues, tryptophan, tyrosine, and occasionally histidine and phenylalanine, are often involved in the binding of the type A C B M s to cellulose (12, 14, 27, 66, 85, 94, 97, 130, 141). These residues are commonly involved in protein-carbohydrate interactions(95, 100, 131, 135). 1.3.2.1 Family 1 Among the first C B M s to be described were those from the Trichoderma reesei cellobiohydrolases C B H I (TrCel7A) and C B H I I (TrCel6A)(128). With the exception of a polysaccharide-binding protein from the alga Porphrya purpurea (GenBank accession U08843), family 1 C B M s are derived entirely from fungal hydrolases. They are the smallest C B M s , typically comprising about 35 amino acids, and having a predominantly P-sheet structure with three conserved tryptophan or tyrosine residues forming a linear platform on one face of the polypeptide. The solution structures of T r C e l 6 A - C B M l (CBD-CbhI)(66) and T r C e l 7 B - C B M l (CBD(EGI))(76) were solved by N M R . Family 1 C B M s bind to insoluble cellulose with an affinity in the range of 105 M " 1 . They also bind to chitin, and interact weakly with cello-oligosaccharides (76, 77). T r C e l 7 A - C B M l (Figure 1.4-A) binds to bacterial micro-crystalline cellulose ( B M C C ) reversibly (73). In contrast, T r C e l 6 A - C B M l 7 (CBDCbhl l ) binds irreversibly to cellulose(17, 91). Mutation of the three surface tyrosines of T r C e l 7 A - C B M l significantly reduced its affinity for insoluble cellulose(72, 97), however the extent of affinity reduction was not quantified. The exposed aromatics of other family 1 C B M s are, presumably, also essential in binding to insoluble cellulose. 1.3.2.2 Family 2a Family 2a is, by far, the most extensively studied family of type A binding modules. Currently, it comprises 54 members, most of which come from bacterial sources, but some are from nematodes (29, 115). Family 2a C B M s comprise approximately 100 amino acids. They bind to crystalline cellulose, including bacterial microcrystalline cellulose ( B M C C ) , and also to partially crystalline preparations, such as phosphoric acid-swollen cellulose (PASC)(88) with an affinity of 1 - 3 x 106 M " 1 (this work and unpublished data). It is not clear, however, i f they have significant affinity for the amorphous regions of phosphoric acid-swollwen cellulose (PASC) . A solution structure of C f X y n l O A - C B M 2 a (hereafter abbreviated to C B M 2 a ) was solved (140). It has a p-barrel type backbone with a relatively flat face on which there are several conserved tryptophan residues (Figure 1.3) involved in binding to cellulose (Figure 1.4-B). It is likely that other family 2a members have a very similar tertiary structure. The involvement of the conserved aromatic residues in binding to cellulose was clearly demonstrated for several family 2a C B M s , including the Cellulomonas fimi modules C e l 6 A - C B M 2 a (formerly CBD C e n A ) (27 ) and C f X y n l O A - C B M 2 a (formerly CBD C e x ) (12) , and Pseudomonas cellulosa X y n l O A - C B M 2 a (85, 94). Binding of C f X y n l O A - C B M 2 a to either B M C C or P A S C is apparently irreversible. Complete removal of the unadsorbed molecules from the solution phase does not lead to significant desorption of bound molecules over extended (i.e., several weeks) periods of 8 equilibration ((25, 57) and B . W . McLean, unpublished data). Although binding is irreversible, the interaction with the cellulose surface is dynamic. Surface diffusion measurements using fluorescence recovery techniques show that C B M 2 a , either in its isolated form or as a module in xylanase 10A, is mobile on the surface of crystalline cellulose (57), and may therefore provide bound xylanase 10A access to a much broader field of substrate. The rate of oxidation of the three conserved surface tryptophan residues of C B M 2 a (W17, W54 and W72) with N-bromosuccinimide is only halved when C B M 2 a is bound to B M C C , suggesting a dynamic interaction of the tryptophans with the cellulose surface which, at least periodically, allows each residue to become solvent exposed and susceptible to oxidation. Furthermore, complete oxidation of the three tryptophan residues eliminates binding but not proper folding of C B M 2 a , indicating the importance of these hydrophobic residues to the binding interaction (12). The thermodynamic behaviour of the binding of C B M 2 a to B M C C is different from that of the typical protein-carbohydrate interaction. Binding is entropically driven. The favourable enthalpy (AH) of binding is quite small making only a small contribution to the overall energetics relative to the change in entropy (TAS). The thermodynamics together with a large negative heat capacity change (AC p ) are consistent with the binding being driven by dehydration of the sorbent and the protein surfaces, specifically the tryptophan-rich binding face(25). The individual contributions of W17, W54, and W72 to the overall binding interaction however, are unknown. It is also unclear whether any other solvent-exposed amino-acid residues proximal to the three tryptophans make a significant contribution to the energetics of binding. CfiCel6B CfiCel48A CfiCel5A CfiCel9A CfiXynlOA CfiCel6A S A . N A . . A T S G G P Q T GG S . P T T G S . T S T P S . S G P A G . . . APGHRQD|Y|AVT[N|QE1PJ I Y T A . N G |V Y S T . N S L W G V . N Q | T J S jTAM HEIV A)NQT| T f ITl LOT I i f NGT T GT TGT TGT TSS ITEiLGD A A L N T T I N I S L S T P L T A P V D . P V S JG r A DO j T A t r p : rp sC rp i JITIY T AC K V S K V Q l * 3LE SAE SATW| SAR S AT W] SST NGTAl T A S T as s T T | s G TAKN TATN TATN TAT G |TVRN S VT S LG . TR . VG . GTV Figure 1.3 Alignment of family 2a C B M s from C.fimi. Each sequence is designated by its enzyme source. Strictly conserved residues are shaded in black, residues having greater than 50% sequence identity are in bold-faced type, boxed and unshaded. 1.3.2.3 Family 3 Family 3 C B M s are common in the non-catalytic scaffoldin proteins of the large, multi-component cellulosome complexes that are produced, most commonly, by cellulolytic, anaerobic bacteria. In addition to a C B M , scaffoldins contain dockerin modules that bind to the cohesin modules of the component cellulases. The C B M of the scaffoldin ensures close interaction between the insoluble target substrates and the associated cellulosome. Family 3 C B M s are the largest of the type A C B M s , usually containing -150 amino acids (6) and bind to insoluble cellulose with an affinity of ~10 6 M " 1 ((41, 84) and this work), similar to that of C B M 2 a . Two C B M 3 s have been characterized in some detail: one from the mesophilic Clostridium cellulovorans, the C B M from cellulose-binding protein A ( C c C b p A - C B M 3 ; formerly designated C B D c l o s hereafter abbreviated C B M 3 ) (40, 41) and another from the thermophilic C. thermocellum, the C B M from cellulose-binding protein A ( C t C i p A - C B M 3 ; formerly Cip-CBD)(84). 8 O CfiCel6B C£iCel48A CfiColSA CfiCel9A CfiXynlOA CfiCel6A L A A| L A P L A P IQP I PA| I S I S S V QT V D V aASVEI SQSTDI I G T A Q F P T B G T A l S F 10 Three crystal structures of three family 3 C B M s are known. Two of them bind to insoluble cellulose: C t C i p A - C B M 3 (Figure 1.4-C) (130) and a similar C B M from the mesophilic Clostridium cellulolyticum ( C c e l C i p C - C B M 3 ; formerly CipC-CBD)(109) . Their structures are somewhat similar to that of C B M 2 a , consisting of two antiparallel (3-sheets. Both of the family 3 C B M s have a calcium ion binding site. Residues that are conserved among family 3 members map to two regions of these C B M s : a shallow groove of underdetermined function on the top of the molecule, and a second area on a flat face on the opposite side of the molecule thought to be the region that interacts with insoluble cellulose. The flat face has conserved tyrosine, tryptophan and histidine residues that are likely the primary residues involved in the binding interaction. In addition, there are residues on this face that could form hydrogen-bonds with the cellulose surface. The role of each of these conserved residues is under investigation. The third C B M 3 structure is from Thermomonospora fusca cellulase 9A (previously E4) (103). It is an internal C B M , flanked by the catalytic domain and a family 2a C B M . It does not bind to crystalline cellulose nor does it have the conserved residues that are implicated in the binding of other C B M 3 s to cellulose. The role of T f C e l 9 A - C B M 3 may be to guide a cellulose molecule into the active site of the catalytic module (103) while the C B M 2 a anchors the enzyme to the insoluble substrate. 1.3.2.4 Family 5 One structure was solved for the 62 amino acid long family 5 C B M from Erwinia crysanthemi Ce l5A (formerly EGZ)(14); it is shaped like a ski boot. Two tryptophans and one tyrosine on one face of the molecule (Figure 1.4-D) are required for effective binding to insoluble cellulose(l 11). One of these tryptophans is also required for secretion of Cel5A(19) 11 1.3.2.5 Family 10 The best studied family 10 binding module is at the N-terminus of Pseudomonas cellulosa xylananase 10A ( P c X y n l O A - C B M l O ) . It comprises 45 amino acids and has similar binding specificity for crystalline cellulose preparations as C B M 2 a . However, it binds with an association affinity constant of 2.5 x 105 M ' 1 , approximately 6 times lower than that of C B M 2 a for similar substrates (39). Its solution structure comprises two anti-parallel P-sheets and a short cx-helix (96). L ike other type A C B M s , C B M 10 has a number of tyrosine and tryptophans, that are conserved within the family, three of which form a platform of aromatic residues along one face of the polypeptide (highlighted in Figure 1.4-E). Two tryptophans and the single tyrosine forming this platform are instrumental in mediating binding to insoluble cellulose (93). Investigation of the thermodynamic roles of other conserved residues located on the binding face is in progress (H.J. Gilbert, personal communication). Type A CBMs Figure 1.4 Structures of type A C B M s . A ) T. reesei cellulase 7 A - C B M 1 B) C.fimi xylanase 1 0 A - C B M 2 a C) C. thermocellum C i p A - C B M 3 D) E. crysanthemi cellulase 5 A - C B M 5 E) P. cellulosa xylanase 1 0 A - C B M 1 0 . Ribbons indicate secondary structure; residues likely to be involved in binding are rendered as "balls and sticks". A l l figures were prepared using M O L M O L (63). 13 1.3.3 T y p e B C B M s Type B C B M s may be thought of as "chain binders". They bind preferentially to single oligosaccharides longer than three monosaccharide units and to soluble polymers. They may also bind to insoluble substrates, such as P A S C , probably to accessible single cellulose chains in these preparations. The binding of this type of C B M is both structurally and thermodynamically similar to the classic protein-carbohydrate interactions exhibited by lectins. Type B C B M s (Figure 1.5) include families 2b, 4, 17, and the recently reclassified family 22 (20). Representatives from families 2b, 4 and 17 wi l l be discussed in more detail here. 13.3.1 Family 2b The seven members of family 2b share amino acid sequence similarity with the C B M s in family 2a. The internal C B M 2 b from C.fimi xylanase 11A ( C f X y n l l A - C B M 2 b - l , hereafter C B M 2 b - l ; Figure 1.5-A), is a typical family 2b C B M . It binds to soluble xylan, with an affinity of ~ 1 0 3 M " \ Despite the obvious amino acid sequence similarity with C B M 2 a (26% identity 39% similarity with two gaps comprising 14% of the aligned region) it does not bind to crystalline cellulose. Structurally, members of families 2a and 2b are strikingly similar except for the positions of the surface tryptophans. In C B M 2 a , the surface tryptophans are in line and coplanar, forming a flat hydrophobic ridge on one face of the molecule; in C B M 2 b - l the tryptophan corresponding to W17 of C B M 2 a is orthogonal to the face of the protein and, with the tryptophan corresponding to W54 of C B M 2 a , forms a twisted binding site for the accommodation of a helical xylan polysaccharide chain (112). C B M 2 b - l lacks a tryptophan corresponding to W72 of C B M 2 a . When the position of the tryptophan corresponding to W17 of C B M 2 a was altered by mutating an adjacent residue, so that it lay parallel to the face 14 of the polypeptide, the mutant bound to crystalline cellulose but had no appreciable affinity for xylan(l 13). The tryptophan residues of family 2 C B M s therefore determine both the affinity and the specificity of binding. 1.3.3.2 Family 4 The N-terminal family 4 C B M from C. fimi Cel9B (CenC) is the most thoroughly characterized type B C B M . This C B M , C f C e l 9 B - C B M 4 - l (formerly C B D N I , hereafter abbreviated C B M 4 - 1 ; Figure 1.5-B) is the N-terminal repeat of the tandem family 4 C B M s of Cel9B. It was the first C B M observed to bind cellooligsaccharides, soluble cellulose derivatives, and the soluble glucopyranoside polymer, barley (3-glucan, with significant affinity ( K a ~10 5 M" 1) (61, 125). It binds to insoluble cellulose preparations with significant amorphous content but not to crystalline celluloses, such as B M C C (23). Solution structures were solved for C B M 4 - 1 (60) and its adjacent module C f C e l 9 B - C B M 4 - 2 (formerly C B D N 2 ; Figure 1.5-C) (13), in the presence of saturating amounts of cellotetraose and cellopentaose, respectively. The two C B M s share 34% amino acid sequence identity and they have very similar structures. Both comprise two antiparallel (3-sheets folded into a jelly-roll sandwich with an obvious groove or cleft on one side. In both C B M s , the cleft is lined by a strip of non-polar amino acids and flanked by hydrogen-bonding residues. The tyrosines in the groove of C B M 4 - 1 are essential for tight binding of insoluble and soluble substrates (64). C B M 4 - 1 may bind the sugar chain in either orientation (58), reflecting the symmetry of the residues in the binding cleft. The crystal structure of C B M 4 - 1 in the presence of cellopentaose confirms the positioning of the substrate within the binding cleft and the specific protein-carbohydrate interactions (A. Boraston, V Notenboom and A . Freelove, personal communication/unpublished results). 15 The binding of C B M 4 - 1 and C B M 4 - 2 is enthalpically driven, consistent with structural evidence for the formation of favorable hydrogen bonds with the equatorial hydroxyls of the cellulose oligomer. C B M 4 - 1 binds a calcium ion at a site located away from binding groove (59). The binding of calcium does not influence the binding of ligands but more likely stabilizes the module. The two modules together in the full enzyme are not separated by any obvious linker region. The affinity of C B M 4 - 1 . 2 for amorphous cellulose is roughly the sum of the affinities of each module, indicating that the binding is not cooperative (13). 1.3.3.4 Family 17 Family 17 C B M s are typified by the 200 amino acid module at the C-terminus of the non-cellulosomal C. cellulovorans cellulase 5 A (formerly endoglucanase EngF), CcCe l5A-C B M 1 7 (hereafter C B M 1 7 ; Figure 1.5-D). It binds to insoluble cellulose (8, 56, 108), cello-oligosaccharides and soluble cellulose derivatives, such as hydroxyethyl-cellulose (HEC) and ethylhyroxyethyl-cellulose (EHEC) . C B M 17 binds soluble cello-oligosaccharides, with the greatest affinity for cellohexaose ( K a = 1.5 x 105 M"'). Overall, C B M 1 7 has similar ligand specificity to C B M 4 - 1 , however, C B M 17 binds with an affinity approximately twice that of C B M 4 - 1 . Even though C B M 17 and C B M 4 - 1 have similar affinities for cello-oligosaccharides comprised of six or five glucose units, respectively, P A S C has approximately three times the binding capacity for C B M 1 7 than C B M 4 - 1 ((8) and this work). It is not known if C B M 17 and C B M 4 - 1 recognize identical regions of the amorphous cellulose in P A S C and this wi l l be investigated. 16 Figure 1.5 Structures of type B C B M s . A ) C.fimi xylanase 11A C B M 2 b - l B) C.fimi cellulase 9 B - C B M 4 - 1 C) C.fimi cellulase 9 B - C B M 4 - 2 D) C. cellulovorans cellulase 5 A - C B M 1 7 E) C. thermocellum xylanase 10B-CBM22-2. Ribbons indicate secondary structure; residues likely to be involved in binding are rendered as "balls and sticks". A l l figures were prepared using M O L M O L (63). 17 1.3.4 T y p e C C B M s Type C C B M s ("small sugar binders") are characterized by their ability to bind simple sugars and consequently, insoluble and soluble polysaccharides (Figure 1.6). These binding modules come from a variety of sources including animals, plants, crustaceans and microbes. They all bind to mono- and disaccharides with significant affinity. They also bind to insoluble and soluble polysaccharide chains and are often desorbed from these matrices by small soluble sugars. C B M s in families 6 (102), 9 (137), and 13 (10) are all type C. C B M family 9 is discussed in more detail below. 1.3.4.1 Family 9 The second, C-terminal family 9 binding module from xylanase 10A of the hyperthermophilic bacterium Thermotoga maritima ( T m X y n l 0 A - C B M 9 - 2 , hereafter abbreviated to C B M 9 - 2 ) is comprised of -170 amino acids. It is unique among the C B M s characterized to date in that it binds strictly to the reducing ends of sugars. C B M 9 - 2 adsorbs to insoluble polysaccharides with an affinity of 1-3 x 106 M " 1 ; it binds to other soluble mono-and soluble polysaccharides with affinities that range from 103 M " ' to 106 M " 1 . Cello-oligosaccharides longer than cellobiose do not bind with any greater affinity, indicating that the preferred ligand is a disaccharide. The crystal structures of C B M 9 - 2 alone (Figure 1.6-A), and in complexes with glucose and cellobiose, show that the binding site is a shallow, blind cleft in which there are two tryptophans with the planes of their ring faces parallel to each other, roughly perpendicular to the surface of the protein. The tryptophans form the entrance to the binding cleft and, when bound to ligand, sandwich the disaccharide. Binding of the substrate is further stabilized by a network of hydrogen bonds involving a number of adjacent charged side-chains. A s with other lectins (75) and C B M s with affinity for soluble sugar ligands, binding to small soluble ligands and cellulose is exothermic, dominated by favourable enthalpy with unfavourable changes in entropy (A. B . Boraston et al; V . Notenboom et al., in press) TypeC CBMs Figure 1.6 Structures of type C C B M s . A ) Thermotoga maritima xylanase 1 0 A - C B M 9 - 2 B) Streptomyces lividans xylanase 1 0 A - C B M 1 3 . Ribbons indicate secondary structure; residues likely to be involved in binding are rendered as "balls and sticks". A l l figures were prepared using M O L M O L (63). 20 1.3.5 Applications of CBMs C B M s have a number of applications that take advantage of their ability to bind to cellulose, which is an inexpensive, abundant and inert matrix. C B M s can be used as affinity tags for protein purification (124, 126). Because it can be desorbed from cellulose using relatively inexpensive sugars, such as glucose, C B M 9 - 2 is especially useful as an affinity purification tag. The irreversible binding of C B M 2 a allows it to be used for the immobilization of processing enzymes, such as the glycosidases, EndoF, and PNGaseF ( A . B . Boraston et al, manuscript in preparation), (3-glucosidase (87) and factor X a (44). The relatively small, compact structures of C B M s make them good scaffolds for the display of libraries of combinatorial peptides (114, 117). 1.5 Goal of this research The goal of this research is to further elucidate the nature of the interaction of C B M s with their ligand. In nature, cellulose forms a myriad of complex structures and is intimately associated with other polysaccharides. Similarly, as discussed in the previous sections, C B M s have an array of structures and a number of different binding specificities. The ligand targeted by each C B M in a complex substrate, composed of a mixture substrate structures, is not precisely known. In this study, the binding specificities of a number of C B M s for the complex cellulose P A S C wi l l be determined by a series of pair-wise competition binding experiments. These experiments wi l l determine the type of cellulose (i.e. crystalline vs. amorphous) bound by each C B M ; currently, there is no method for directly evaluating the binding specificity of C B M s for insoluble substrates, particularly complex substrates such as P A S C . Overall, these experiments wi l l demonstrate that the diversity of C B M structure and 21 function is merely a reflection of the diversity of structures that cellulose and cellulose-associated polysaccharides can, and do, adopt. Because of its prominence in the C.fimi cellulase system and its potential applications, the interaction and the binding of C B M 2 a to cellulose wi l l be investigated in more detail with a goal of working towards the formulation a functional and structural model of binding to insoluble cellulose. C B M 2 a binds to cellulose with apparent irreversibility; its ability for bound molecules to exchange with molecules in the solution phase, as demonstrated with other cellulases, wi l l be investigated. C B M 2 a has a number of conserved residues on the binding face, including three tryptophans that have been implicated in the binding reaction. Site-directed mutation and a Langmuir-type adsorption isotherm analysis wi l l be used to determine the individual energetic contributions of W17, W54, W72 and a number of neighbouring residues to the overall binding affinity of C B M 2 a for cellulose. Structural information regarding C B M s bound to the surface of insoluble cellulose is difficult to obtain using solution-based methods, such as liquids N M R . This study wi l l work towards a structural view of C B M 2 a bound to the surface cellulose by the use of solid state N M R ; the first attempt to directly observe a C B M bound to insoluble cellulose. In the future, this work wi l l contribute towards a structural and functional model of the binding interaction between C B M 2 a and the surface of insoluble cellulose. 22 2. Materials and methods 2.1 Materials and reagents A l l chemicals were of analytical or high pressure liquid chromatography ( H P L C ) grade, and puchased from Sigma (St. Louis, M O ) , B D H (Toronto, ON) , or I C N (Aurora, OH) , unless otherwise noted. Solutions were prepared as described by Sambrook et al. (104). Avicel® PH-101 was from F M C International (Cork, Ireland). Bacterial microcrystalline cellulose ( B M C C ) and phosphoric acid-swollen cellulose (PASC; prepared from Avicel® P H -101) were prepared by Emily Kwan, as described previously (37, 52, 139). 2.2 Bacterial strains and plasmids 2.2.1 Escherichia colistrains and plasmids C B M 2 a and its mutants were produced using the Escherichia coli strains JM101 (142), R1360, and BL21(DE3) (120) (Novagen, Inc.; Madison, WI). The expression vector used for production of C B M 2 a and its mutants, p T u g K - H 6 - f E G R - C B M 2 a , is a derivative of the pTugA and pTugAS vectors described previously (43). It encodes resistance to kanamycin; it also carries a gene fragment encoding the leader peptide of X y n l O A at the N-terminus, followed by six histidine residues, a factor X a protease cleavage site (amino acid sequence, IEGR), and then a synthetic gene fragment encoding C B M 2 a at the C-terminus (Figure 2.1). 23 C B M 2 a BstBI Figure2.1 Restriction map of the CBM2a expression vector pTugK-H6-IEGR-CBM2a. This vector has a pUC origin of replication (pUC Ori), encodes for kanamycin resistance (KmR), and the Lac repressor. It has a gene fragment encoding the leader peptide of XynlOA at the N-terminus, followed by six histidine residues, a factor Xa protease cleavage site (amino acid sequence, IEGR), and then a synthetic gene fragment encoding CBM2a at the C-terminus. Only unique restriction endonuclease recognition sites are indicated. 2.2.2 Acetobacter xylinum strains A. xylinum strains 53524, 23769, 100821, and 700178 were obtained from the American Type Culture Collection (ATCC; http://www.atcc.org) and from laboratory stocks maintained by Emily Kwan. 2.3 Culture media 2.3.1 E. coli growth media E. coli were cultured in tryptone, yeast extract, phosphate medium (TYP) (104) or M9 minimal medium. M9 minimal medium was prepared as follows: a solution containing 6 g of Na 2HP0 4, 3 g of KH 2 P0 4 , 0.5 g NaCl, and 1.0 g of NH4C1 made up to 100 ml with dH20 was 24 sterilized and added to 871 ml of sterile dH 2 0. The following components, each dissolved in dH 2 0 and sterilized separately, were then added: 2 ml of 1 M M g S 0 4 , 1 ml of 0.01 M F e C l 3 , 1 ml of 1 mg/ml thiamine and 25 ml of 40% (w/v) glucose. 2.3.2 Acetobacter xylinum growth m e d i a Two media were used to culture A. xylinum in the production of bacterial cellulose: a rich undefined medium and a defined minimal medium. One litre of buffered H & S medium (53), the rich medium, was prepared as follows: 5 g of yeast extract, 5 g of peptone, 2.7 g of sodium phosphate and 1.15 g of citrate were dissolved in 950 ml of dH 2 0, adjusted to p H 5.0 with HC1 and then sterilized. 50 ml of 40% (w/v) glucose (in dH 2 0, sterilized separately) were then added. One litre of the minimal medium (31) was prepared as follows: a solution was made (Stock Solution A ) containing 1.15 g of citric acid, 1.0 g NH 4 C1, 2.7 g N a 2 H P 0 4 , and 0.1 g K C I made up to 100 ml with dH 2 0, and sterilized. The defined medium was made by adding Stock Solution A , 10 ml of MgSO 4 -7H 2 0 (25-g/liter stock solution prepared and sterilized separately), 25 ml of 40% (w/v) glucose (filter sterilized) and 1 ml of sterile 7.5 mg/ml stock solution of niacin to 864 ml of sterile dH 2 0 for a final volume of one litre. 2.4 DNA manipulations A l l D N A manipulations were performed by standard methods as described in Sambrook et al. (104). Restriction endonucleases and T4 D N A ligase were purchased from New England Biolabs ( N E B ; Beverly, M A ) or Life Technologies Canada (Burlington, ON) , and used in the buffers provided by the manufacturers according to their directions. 25 2.4.1 Synthesis of DNA fragment encoding CBM2a The D N A fragment encoding C B M 2 a was synthesized by a PCR-based procedure, similar to that used in the synthesis of a xylanase gene from Schizophyllum commune (42). The sequence incorporated a number of unique restriction sites to facilitate manipulation of the fragment after site-directed mutation (Figure 2.1), and the codon bias was changed to that of E. coli, both without changing the encoded amino acid sequence. Six oligonucleotides, synthesized by the U B C Nucleic A c i d and Protein Services Unit (NAPS) , were used as primers in the P C R assembly of the complete fragment (Table 2.1). The synthesis was performed in two steps, as outlined in the schematic presented in Figure 2.2. Table 2.1 Oligonucleotides used for the construction of the synthetic H i s 6 - C B M 2 a gene fragment. Primer Oligonucleotide Sequence S C x l 5 ' G A C T A G A A T T C A G G A G G A A A C A G C T A T G C C G C G T A C T A C A C C A G C - 3 ' SCx2 5 A T G C C G C G T A C T A C A C C A G C T C C G G G T C A C C C G G C T C G T G G T G C T C G T A C C G C T C T G C G T A C C A C C C G A C G T C G T G C T G C T A C C C T G G T T G T T G G T G C T A C C G T T G T T C T G C C A G C T C A G G C T G C T A G C G G T C C A G C C G G C T G C C A G G T T C T G T G G G G T G - 3 ' SCx3 5 ' A C C T G C T G A C C C G A G G G A A A A G A G A A G G T C A G G G T C C A A C C G T C A A C C G G A G C A G A G C T C G T G T T T T T A A C G G T A A C G T T A G C G G T G A A A C C G G T G T T C C A C T G G T T A A C A C C C C A C A G A A C C T G G C A G - 3 ' SCx4 5 ' T A G A T G A C C A A G C C T G A G T T A C C T G C T G A C C C G A G G G A A A - 3 ' SCx5 5 ' T G C A A G G G G T A C C G T T C A G A G A G A A A G C G G T T G G A G C A G C G T T G G T A C C G G T G T G A G A A C C G T T G A A A C C G A A C T G A G C G G T T C C A C C T G C A G G G A T A G A A C C G T T C C A C G G A G C G T T A C G A A C T G T A A C A G C G G A T C C A G A C T G G G T A A C T G T A G A T G A C C A A G C C T G A G T T - 3 ' SCx6 5 ' G T T A C A A G C T T T T A T T A A C C A A C G G T G C A A G G G G T A C C G T T C A G A - 3 ' 26 A) S C X L » SCx2 ^ | SCx3 vl/ * SCx4 SCx(2/3) S C x l ^ SCx5 SCx(2/3) 4 SCx6 CBM2a F i g u r e 2.2 Schematic for the two-step construction of the H i s 6 - C B M 2 a fragment. The first step (A) resulted in a D N A fragment of approximately two-thirds the length (SCx(2/3)) of the final D N A fragment. SCx(2/3) was used in a second P C R reaction (B) resulting in the full length product that was cloned into pUC19 and pTugK The first reaction mixture contained 5 pmol each of longer primers SCx2 and SCx3, 200 u M of each deoxynucleoside triphosphate (dNTP), 3 U of Expand H i - F i Polymerase (Roche, Mannheim, Germany), and its recommended buffer supplemented with D M S O to 5%, in a final volume of 50 p L . After five cycles of 1 min at 95°C followed by 3 min at 72°C in a Perkin-Elmer model 2400 Thermocycler, 20 pmol each of the flanking primers S C x l and SCx4 were added. Synthesis of the segment was completed by 25 cycles of 45 s at 95°C, 45 s at 58°C, and 1 min at 72°C. The second reaction mixture contained 1 ng of the product from the first reaction, 23.00, 2.30, or 0.23 pmol of primer SCx5 (all three primer concentrations yielded P C R product), 2.5 U of Pwo polymerase in its recommended buffer supplemented with D M S O to 5% (v/v), and 200 u M of each dNTP, in a final volume of 50 p L . After five cycles of 1 min at 95°C followed by 3 min at 72°C, 20 pmol each of the flanking primers S C x l and SCx6 were added and synthesis of the fragment completed by twenty-five cycles of 45 s at 95°C, 45 s at 55°C, and 1 min at 72°C. The full length fragment was purified using the Qiaquick P C R purification kit (Qiagen Inc., Chatsworth C A ) , digested with EcoRI and 27 Hindlil, then ligated into pUC19 (142) that had been digested with the same enzymes. After checking its sequence, the fragment was subcloned into the pTugK vector. 2.4.2 Construction of CBM2a mutants A l l mutations were made by two-primer P C R mutagenesis (1) or the 'megaprimer' method (106, 116). A typical two primer reaction contained 50 ng of template D N A , 5 pmol each of the flanking (primer B G 2 , Table 2.2) and the mutagenic primers (Table 2.2), 200 p M of each dNTP, 1 U of Vent R Polymerase (NEB) in its recommended buffer. Amplification was obtained by 30 cycles of 30 s at 96°C, 30 s at 53°C and 60 s at 72°C. The product was purified with a Qiaquick P C R purification kit (Qiagen Inc.), digested with the appropriate restriction enzymes, and ligated into a pTugK vector that had been digested with the same enzymes. Mutants of residues W17, W54, W72 and Q52 were made by this method. Mutants of residues N15, N18, Q83, and N87 were made by using a megaprimer strategy. The first product was amplified using the mutagenic primer and the B G 2 flanking primer as described above. The product of this reaction was used as a megaprimer using the oligonucleotide Lax 16 as a flanking primer. P C R conditions were as described previously (116). Briefly, 2 ug of megaprimer product were used with 50 ng template (pTugK-H6-IEGR-CBM2a) and 5 pmol of the Lax 16 flanking primer, added after 5 cycles of denaturation at 96°C for one min, and extension at 72°C for three min. The remaining 30 cycles of the P C R program were as described above. 28 Table 2.2 Oligonucleotides used for the construction of H i s 6 - C B M 2 a mutants Primer Ol igonucleotide s e q u e n c e 3 W 1 7 Y 5 ' T G G G G T G G T A C C C A G T A T A A C A C T G G T T T C A C C G C T A A C 3 ' W17F 5 ' T G G G G T G G T A C C C A G T T T A A C A C T G G T T T C A C C G C T A A C 3 ' W 1 7 A 5 ' T G G G G T G G T A C C C A G G C G A A C A C T G G T T T C A C C G C T A A C 3 ' W 5 4 Y 5 ' T T T C C C T C G G G T C A G C A G G T A A C T C A G G C T T A T T C G T C T A C A G T T A C C C A G 3 ' W54F 5 ' T T T C C C T C G G G T C A G C A G G T A A C T C A G G C T T T T T C G T C T A C A G T T A C C C A G 3 ' W 5 4 A 5 ' T T T C C C T C G G G T C A G C A G G T A A C T C A G G C T G C G T C G T C T A C A G T T A C C C A G 3 ' W 7 2 Y 5 ' C A G T C T G G A T C C G C T G T T A C C G T A C G T A A C G C T C C G T A T A A C G G T T C T A T C C C T 3 ' W72F 5 ' C A G T C T G G A T C C G C T G T T A C C G T A C G T A A C G C T C C G T T T A A C G G T T C T A T C C C T 3 ' W 7 2 A 5 ' C A G T C T G G A T C C G C T G T T A C C G T A C G T A A C G C T C C G G C G A A C G G T T C T A T C C C T 3 ' N 1 5 A 5 ' G G C T G C A C C G T A C T G T G G G G T G T T G C C C A G T G G A A C A C T G G T T T G A C C G C T 3 ' N 1 8 A 5 ' G G C T G C A C C G T A C T G T G G G G T G T T A A C C A G T G G G C C A C T G G T T T G A C C G C T 3 ' Q52A 5 ' T T T C C C T C G G G T C A G C A G G T A A C T G C G G C T T G G T C G T C T A C A G T T A C C C A G 3 ' Q83A 5 ' T C T A T C C C T G C G G G T G G A A C C G C T G C G T T C G G T T T C 3 ' N 8 7 A 5 ' G G T T G G C G C C G C G T T G G T A C C G G T G T G A G A A C C G G C G A A A C C G A A C T G 3 ' N 8 7 H 5 ' G G T T G G C G C C G C G T T G G T A C C G G T G T G A G A A C C G T G G A A A C C G A A C T G 3 ' Lax 16 5 ' T A G G A C C A C G C C C G C A 3 ' B G 2 5 ' T G A T C A G A T C T T G A T C C C C T G 3 ' "underlined nucleotides indicate the loci of changes made for amino acid substitution or introduction of a restriction endonuclease recognition site 2.5 Production and purification of CBMs 2.5.1 Production of C B M s used in competition isotherms Representative C B M s from a number of different C B M families were used in the competition binding studies (Table 2.3). A l l of these C B M s were produced recombinantly and provided from a number of sources, as indicated. The molar extinction coefficients, calculated from aromatic amino acid content by the method of Pace and co-workers, are also indicated (90). 29 Table 2.3 C B M s used in competition isotherms on P A S C C B M B io log ica l S o u r c e Ext inct ion C o e f f i c i e n t (e28o: M"1-cm"1) S o u r c e C f C B M 2 a - X y n l O A C.fimi Xylanase 10A (Cex) 27 625 a C f C B M 2 a - C e l 6 A C. fimi Cellulase 6 A (CenA) 36 105 b C c C B M 3 - C b p A C. cellulovorans Cellulose-binding 24 300 c protein A ( C B D c l 0 S ) C f C B M 4 - l - C e l 9 B C.fimi Cellulase 9B (CenC) 21 370 d C c C B M 1 7 - C e l 5 A C. cellulovorans Cel5A(EngF) 31 310 e T m C B M 9 - 2 - X y n l O A T. maritima Xylanase 10A (XynA) 43 430 e a provided by P h i l i p Wendler and Roberta Farrel l , ;Sandoz Chemicals Biotech Research Corporation; b prepared by Dr . Ken Tokayasu; c provided by C B D Technologies, Rehovot Israel; d prepared by E m i l y K w a n and E m i l y Amandoron-A k o w ; e provided by Dr . Al i sda i r Boraston and Patrick C h i u 2.5.2 P r o d u c t i o n a n d purif ication of C B M 2 a a n d mutan ts The full-length fragment was ligated into the vector pTugK in frame. Inclusion of the leader peptide of X y n l O A in the fragment allows export of C B M 2 a to the periplasm of E. coli from where it leaks into the culture supernatant (42, 88). Since the aim was to obtain mutants of C B M 2 a with reduced affinity for cellulose, a hexahistidine sequence followed by a factor X a site was added to the N-terminus of mature C B M 2 a so that the mutant polypeptides could be purified by immobilized metal-chelate affinity chromatography ( I M A C ) (92). This had the added advantage of avoiding the denaturing conditions required to desorb C B M 2 a from cellulose if the mutants, unlike the wild-type, could not be refolded following desorption (88). In the event that the hexahistidine sequence affected the binding of C B M 2 a to cellulose, factor X a could be used to obtain native C B M 2 a following purification by I M A C . E. coli strains JM101 or R1360 were used for the production of wild-type C B M 2 a and its mutants. Typically, 500 ml of T Y P containing 1.5 m M potassium phosphate and 50 pg kanamycin-ml"1 were inoculated with overnight cultures of an E. coli strain transformed with the appropriate vector. The cultures were shaken at 200 rpm at 37°C until the O D 6 0 0 was -1.0, then induced with I P T G at a final concentration of 0.3 m M . After a further 36 hours, the cells were removed by centrifugation. The proteins in the supernatant were concentrated 30 and exchanged into I M A C binding buffer (5 m M imidazole, 5 M N a C l , 20 m M Tr i s -HCl , p H 7.9) using a tangential flow filtration unit (Filtron Ultrasette, 1 kDa cut-off). The solution was passed through a column of Ni 2 +-Sepharose (His-Bind resin; Novagen, Milwaukee, MI) previously charged with 10 column volumes of 50 m M N i S 0 4 and equilibrated with binding buffer. The column was washed with binding buffer. Adsorbed polypeptide was eluted with binding buffer containing stepwise increases in the concentration of imidazole (Figure 3.1). The fractions containing polypeptide were detected by running approximately 20 pl of each fraction on a sodium dodecyl sulfate polyacrylamide gel electrophoresis ( S D S - P A G E ) gel, then staining with Coomassie brilliant blue (104). Fractions containing C B M 2 a were pooled, desalted and concentrated in a stirred ultrafiltration cell (Amicon; Filtron 1 kDa cut-off filter). Protein purity and approximate yield were estimated by S D S - P A G E . Final protein concentrations were determined from the A 2 8 0 n m , using e 2 8 0 of 27 625 M' -cm" 1 for the wild-type polypeptide, 22 125 M ' c m " 1 for the single tryptophan to alanine or phenylalanine mutants and 23 615 M ' - cm" 1 for single tryptophan to tyrosine mutants (90). The wild-type and mutants of C B M 2 a were obtained routinely in yields of up to 100 mg of purified polypeptide per litre of culture supernatant, except for the W 5 4 A mutant, for which the yield was approximately 20 mg per litre. In each case, sufficient polypeptide was obtained for binding analysis. 2.5.2.1 M9 Media for production ofl5N-CBM2a M 9 minimal medium was used for the production of the 1 5N-labelled form of the C B M 2 a polypeptide, 1 5 N - C B M 2 a . For its production, l g of 1 5 N H 4 C 1 was substituted for the NH 4 C1 in the salts solution. 31 2.5.2.2 Production of15N-labelled CBM2a Five millilitre cultures of T Y P , supplemented with kanamycin to 50 u.g/ml, were inoculated with a single colony of E. coli BL21(DE3) that had been freshly transformed with pTugK-H 6 - I E G R - C B M 2 a . The 5 ml cultures were incubated overnight at 30°C. The next day, the cultures were washed twice by centrifugation and resuspended in 5 ml of M 9 minimal medium. 500 u.1 of the resuspended culture was inoculated into 500 ml of M 9 minimal medium, in which 1 5 N H 4 C 1 was substituted for the NH 4 C1 in the salts solution, supplemented with kanamycin to 50 | lg/ml. The cultures were incubated at 30°C, with shaking. When the A 6 0 0 reached -0.6, after approximately 12 h, the culture was induced by the addition of IPTG to a concentration of 0.3 m M and incubated overnight. Cells were harvested and l 5 N - C B M 2 a was purified from culture supernatant and the cell extract as described in sections 2.5.2. Typically, production yielded 6-10 mg of purified 1 5 N - C B M 2 a per litre of culture. 2.6 Production and processing of bacterial cellulose 2.6.1 P r o d u c t i o n of 1 3 C - e n r i c h e d C e l l u l o s e 2.6.1.1 Production of nC-4-enriched Cellulose Strains of A. xylinum were obtained from the American Type Culture Collection ( A T C C ) and from laboratory stocks. The strains, A T C C numbers 53524, 23769, and 10821, were screened for their ability to grow and produce a substantial cellulose pellicle in the defined medium. In all of the following steps, duplicate cultures of each A. xylinum A T C C strain were inoculated and incubated at 30°C; one culture was shaken at 200 rpm and the other was static. Two 5 ml cultures of H & S medium were inoculated from frozen stocks with each A. xylinum strain. After 5 days, once a visible cellulose pellicle had formed, each culture was shaken briefly on a vortex mixer and 5 | l l of each culture inoculated into 5 ml tubes of A. xylinum minimal 32 medium. Only A T C C strains 53524 and 23769 grew in minimal medium; both strains produced cellulose in shaken and static culture. After 10 days, each culture was briefly shaken on a vortex mixer and 500 pl inoculated into 100 ml of defined medium in 250 ml flasks. After approximately one month, both strains, in both agitated and static conditions, had produced cellulose but A T C C 23769 grown at 30°C without agitation produced the largest amount, measured by weight, after a series of processing steps (see section 2.6.2). To produce l 3C-4-enriched cellulose, 5 ml of defined medium were inoculated with 5 p l of a culture of A T C C 23769, that had been inoculated from frozen stocks. After five days of static incubation at 30°C, 500 pl of this culture were inoculated into each of two 250 ml flasks containing 100 ml of defined medium with 1% D-glucose-4 1 3C (Cambridge Isotope Laboratories, Andover, M A ) . After approximately one month of static incubation at 30°C, the cellulose pellicles were harvested and processed (as described in section 2.6.2). 2.6.1.2 Production ofl,3,4,6-'3C-enriched cellulose The synthesis of cellulose using D-glucose-4 1 3C did not label just the C-4 of the resulting cellulose, there was a certain amount of scrambling of the label, it was decided that a simple enrichment of 1 3 C using the undefined H & S medium and less costly labelled carbon source, glycerol- 1,3- 1 3C 2, would be sufficient to make a larger amount 1 3C-enriched cellulose (30). A. xylinum A T C C strains 100821, 23769, 53524 and 700178 were screened for their ability to grow and produce cellulose in H & S medium containing 1% (w/v) glycerol (hereafter H & S -glycerol) instead of 1% (w/v) glucose, at 30°C without agitation. A l l strains produced cellulose in H&S-glycerol . However, cultures of A T C C 53524 produced the most cellulose (191.2 mg from 100 ml of culture). Larger 1 3 C enrichment cultures were prepared as follows: 500 pl of a 5 ml culture of 53524 in H&S-glycerol were inoculated into each of four 125 ml 33 flasks containing 40 ml of H & S containing 0.5% (w/v) glycerol-1,3- 1 3 C 2 (Cambridge Isotope Laboratories, Andover, M A ) and 0.5% glycerol (unlabelled). After 10 days of incubation at 30°C, cellulose pellicles and associated cells were harvested and processed as described in section 2.6.2. 2.6.2 P rocess ing of bacterial cel lulose Cellulose pellicles from 1 3C-enrichment cultures were harvested and processed by a method similar to that described by Hestrin, 1963 (52). The thick cellulose and cell pellicle was removed, blended with 100 ml of 1 M N a O H at "Frappe" in an Osterizer cylco«trol 10 blender (Sunbeam Corporation, Boca Raton F L ) for 1 min, and incubated overnight at room temperature with gentle rocking. The cellulose was then washed extensively with dH 2 0 through a suction filter, rinsed with 200 ml 0.5% acetic acid, then washed with dH 2 0 until the p H of the filtrate was neutral. The bacterial cellulose was stored in dH 2 0 supplemented with sodium azide to 0.02% (w/v). 2.7 Determination of affinities The affinities of C B M s and C B M 2 a mutants for insoluble cellulose were determined using a solution depletion method to generate binding isotherms. C B M s were equilibrated in microcentrifuge tubes with 1 mg of cellulose ( B M C C or P A S C ) in 50 m M potassium phosphate (pH 7.0) in a total reaction volume of 1 ml. In binding experiments with Avicel®, 5 mg of cellulose was used. The final total polypeptide concentrations ranged from 1 p M to 30 p M . Duplicate or triplicate samples were incubated at 4°C for at least three hours while rotating end over end. The cellulose was removed by centrifugation and the concentration of protein remaining in the supernatant, termed the free protein concentration, was determined by A 2 8 0 n m after subtracting A 3 5 0 n m to allow for light scattering. The concentration of bound 34 protein for a particular concentration of the C B M was the difference between a control incubated without cellulose (total protein) and protein remaining in the supernatant in samples after incubation with cellulose (free protein). A n isotherm of [Bound] (fxmol-g"1 of cellulose) vs [Free] (|J.M) was generated and binding parameters were determined by non-linear regression of the Langmuir-type isotherm (Equation 1; modified from (27)), using GraphPad Prism 3.0 for Windows (GraphPad Software Inc., http://www.graphpad.com): [B] = ^CN 0 ] -K a - ( [F ] -G) > | G (1) 1 + K a ( [ F ] - G ) I where [B] is the concentration of bound protein (pjnol-g 1 cellulose), [N 0] is the total concentration of binding sites (u\mol-g~' cellulose), K a is the association affinity constant (fimolT'), [F] is the concentration of free protein (u.mol-f1) and G is a constant, calculated during the regression, that corrects for non-protein absorbance at the measured wavelengths. G therefore is the sum of all of the factors that contribute to absorption measurements not specific to the C B M . 2.8 Competition binding isotherms 2.8.1 L a b e l l i n g of C B M s with f luorescen t p r o b e s A series of C B M preparations was made, each labelled with one of two different fluorescent probes so that in a binding reaction containing more than one C B M , the concentration of each species could be measured irrespective of the A 2 8 0 . Oregon Green® 514 carboxylic acid succinimidyl ester and 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid succinimidyl ester ( A M C A - X , SE) (Molecular Probes, Eugene Oregon) were chosen as the two fluorescent probes because they have distinct excitation and emission characteristics 35 (Figure 3.5). Oregon Green® has an absorbance maximum at 506 nm, with an emission maximum of 537 nm. A M C A - X ( A X ) absorbs maximally at 352 nm with an emission maximum at 447 nm. These succinimidyl ester probes react with the primary amine groups of C B M s , the N-terminus, and solvent-exposed lysine residues, to form a stable covalent linkage. Labelling reactions were performed as directed by the manufacturer. Briefly, 1 mg of the fluorescent probe dissolved in 100 p l of dimethylsulfoxide ( D M S O ) was added to 10 -15 mg of C B M in approximately 1 ml of 0.1 M sodium bicarbonate buffer, p H 8.3. The solution was mixed in the dark, at room temperature, for 1 hr. The labelled polypeptide was passed twice through a 5 ml Sephadex G-25 column (Pharmacia Biotech, Uppsala Sweden), equilibrated with 50 m M potassium phosphate (pH 7.0), to separate any unbound probe from the protein. Fractions containing protein, as detected by A 2 8 0 , were collected and pooled. The labelling efficiency of each reaction was calculated based on the extinction coefficients provided for each label at their maximum absorption wavelength ( 8 3 5 2 for A M C A - X is 18 500 M •cm' ; £ 5 0 6 for Oregon Green® is 86 200 M '•cm*1; Molecular Probes). 2.8.2 S t a n d a r d c u r v e s of f l u o r e s c e n c e v s . p o l y p e p t i d e c o n c e n t r a t i o n Because of the sensitivity of the fluorimeter (Perkin Elmer L S 50 Luminescence Spectrometer), labelled C B M was diluted with unlabelled C B M so that maximum measurable fluorescence emission (1 000 fluorescence units) coincided with typical maximum concentrations of C B M s used in an isotherm, usually 30 p M . The appropriate dilution was determined empirically by making a series of dilutions, measuring the fluorescence of each dilution, and adjusting the excitation and emission slit widths until the measured fluorescence fell within the range measurable by the fluorimeter. For example, this ratio was approximately 373 moles of unlabelled C B M 2 a per mole of Oregon Green®-36 labelled C B M 2 a and 87 moles of C B M 4 - 1 per mole of A M C A - X - l a b e l l e d C B M 4 - 1 . Fluorescence emission at 537 nm of Oregon Green-labelled C B M s was measured for 2.0 s using an excitation wavelength of 506 nm, excitation and emission slits appropriate for each labelled C B M preparation (typically 7.5 nm), and a 530 nm emission filter. Fluorescence emission at 447 nm of A M C A - X - l a b e l l e d C B M s was measured using an excitation wavelength of 352 nm, without an emission filter, with a collection time of 2.0 s at the appropriate excitation and emission slit widths. Conversion of fluorescence to molar protein concentration required a reliable standard curve. Standard curves for fluorescence vs. free and total protein concentration were obtained by performing an isotherm with each fluorescently-labelled C B M preparation. The fluorescence and net A 2 8 0 (the A 2 8 0 corrected for light scattering estimated from the A 3 5 0 ) of each duplicate set of free ( C B M samples equilibrated with cellulose, and centrifuged to separate the supernatant from the cellulose) and total ( C B M samples equilibrated in an equal volume of buffer, without cellulose) samples were measured. The fluorescence of each sample was plotted versus the calculated molar concentration of that sample. The contribution of the fluorescent probe conjugate to the A 2 8 0 n m of the polypeptide solution was negligible. Standard curves were unique to each C B M preparation and each cellulose substrate used. 2.8 .3 Isotherms by f l u o r e s c e n c e The veracity of each standard curve, as well as the effect of the fluorescent probe on binding, was assessed for each labelled C B M preparation by comparing the binding curves and binding parameters obtained by U V absorbance (net A 2 8 0 ) and fluorescence. A standard curve of free and total fluorescence vs. C B M concentration (pM) was constructed from a binding experiment using unblocked microcentrifuge tubes. A second isotherm, using tubes that had 37 been blocked in a solution of 1% bovine serum albumin (BSA) by soaking them for 2 hours then rinsing them dH 2 0, was then performed. The fluorescence of each sample was measured and the molar concentrations of each free and total sample were calculated by interpolation using the appropriate standard curves. The resulting isotherms from U V absorbance and fluorescence measurements were plotted and compared. 2.8 .4 C o m p e t i t i o n i s o t h e r m s In a binding competition experiment consisted of the measurement of the binding of one C B M to cellulose ( B M C C or P A S C ) was measured in the presence of a constant concentration of a second C B M (a competitor). The binding modules used in the competition experiments (Table 2.1) were representatives of different C B M families. In each case, the C B M and competitor were added simultaneously. After equilibration and isolation of the supernatant, the fluorescence of the C B M and its competitor, was measured. The molar concentration of each component was calculated from interpolation using the appropriate standard curves. A l l competition experiments were performed using BSA-blocked microcentrifuge tubes to minimize any non-specific adsorption of the C B M s to the microcentrifuge tubes. In cases where the binding of a C B M , acting as a competitor, was affected by the presence of the fluorescent probe (eg. C B M 9 - 2 ) , an isotherm of one C B M (not affected by being labelled) was performed in the presence of an unlabelled competitor. 2.9 Adsorption and surface exchange of CBM2a to cellulose 2.9.1 S u r f a c e e x c h a n g e of C B M 2 a The extent to which C B M 2 a in solution is able to exchange with binding-modules bound to the surface of B M C C was assessed by an exchange experiment using two preparations of C B M 2 a , one labelled with Oregon Green®, the other with A M C A - X . C B M 2 a - A X , at a total concentration of approximately 20 p M was incubated with 1 mg of B M C C at 4°C for at least three hours in a total sample volume of 1 ml. The cellulose was pelleted by centrifugation and 900 p l of the supernatant were removed. The amount of bound C B M 2 a -A X was calculated from the difference between the molar concentration of samples of equal volume incubated with and without cellulose as measured by A M C A - X fluorescence and interpolated using the appropriate standard. Each B M C C pellet was washed three times with 900 pl of 50 m M potassium phosphate buffer (pH 7.0) by centrifugation and subsequent removal of the supernatant. C B M 2 a - O G was added to the washed pellets at three concentrations: at approximately the measured free equilibrium concentration of C B M 2 a - A X (~4 p M ) , at greater than the equilibrium concentration (-15 p M ) and at less than the equilibrium concentration (-2 p M ) . The pellets were resuspended and incubated for a further three hours. After removal of the cellulose by centrifugation, the fluorescence of C B M 2 a - O G and C B M 2 a - A X was measured. The concentrations of remaining C B M 2 a - A X bound, and C B M 2 a - O G bound were calculated. 2.9.2 K ine t ics of a d s o r p t i o n to P A S C a n d B M C C The rate at which C B M 2 a reaches equilibrium with sites on the surface of insoluble cellulose was measured by incubating samples of C B M 2 a solution with 1 mg of B M C C or P A S C for different times at 4°C. Cellulose was separated from the supernatant by passing the suspension through a Qiaquick spin column (Qiagen Inc.). The fluorescence values of the free and total samples were measured, the molar concentration determined, and the amount of bound C B M calculated. The fraction bound ( C B M 2 a bound at time t ( [BJ divided by the concentration bound at equilibrium ([B]; t>180 min) was plotted versus time (min) and the 39 rate of adsorption was calculated by non-linear regression using equation 2, a one site exponential association model: ^ 1 = ( 1 - ^ ) <2> [B] where [J3t] is the concentration of CJ3M2a bound (umol-g 1 cellulose) at elapsed time t (min), [B] is the concentration of C B M 2 a bound at equilibrium (uxnol-g"1 cellulose), and k is the rate constant. 2.9 .3 K ine t ics of C B M 2 a e x c h a n g e o n B M C C The rate at which C B M 2 a in solution is able to exchange with binding-modules bound to the surface of B M C C was assessed by a kinetic exchange experiment using two preparations of C B M 2 a , one labelled with Oregon Green, the other with A M C A - X . A number of samples were prepared in which C B M 2 a - A X was equilibrated with 1 mg of B M C C at a total concentration of 20 U.M, in a total volume of 1 ml . The cellulose was pelleted by centrifugation and 900 fil of the supernatant were removed. A s with the previously described equilibrium exchange experiments, the amount of bound C B M 2 a - A X was calculated from the difference between the molar concentration of samples of equal volume incubated with and without cellulose as measured by A M C A - X fluorescence and interpolated using the appropriate standard. Each B M C C pellet was washed three times with 900 | l l of 50 m M potassium phosphate buffer (pH 7.0) by centrifugation and subsequent removal of the supernatant. C B M 2 a - O G was added, at a final total concentration of 20 U.M, and incubated for different times at 4°C. Cellulose was separated from the supernatant by passing the suspension through a Qiaquick spin column. The fluorescence of the free and total samples of C B M 2 a - A X and C B M 2 a - O G was measured, the molar concentrations of each determined, and the amount of bound C B M 2 a - A X and C B M 2 a - O G was calculated. 40 2.10 NMR 2.10.1 S o l i d s t a t e N M R : C r o s s - p o l a r i z a t i o n / m a g i c a n g l e s p i n n i n g ( C P / M A S ) Solid state M A S N M R experiments were performed on a Bruker DSX-400 spectrometer using a 4 mm Bruker triple-tuned probe with zirconia rotors and K e l - F caps. The spectrometer operated at frequencies of 400.13 M H z for ' H , 100.61 for 1 3 C , 40.54 for 1 5 N and 376.50 for 1 9 F . Chemical shifts were referenced to tetramethylsilane (TMS) using Adamantane as a secondary reference for I 3 C spectra. The 'H-> 1 3 C C P Hartmann-Hahn match condition was set up using Adamantane; the 'H-> 1 3 C-> 1 5 N match was set up using 1 3 C, 1 5 N-glycine . Unless otherwise specified, all spectra were collected at room temperature on wet samples (40%-60% water content) at a spinning rate of 5 kHz. I 3 C C P / M A S spectra of unlabelled cellulose ( l 3 C , 1.1% natural abundance) were the sum of 40 000 scans, collected using a standard C P pulse sequence, using a 4.5 ps proton 90° pulse, a 1000 ps contact pulse and a 2 s delay between repetitions. 1 3 C C P / M A S spectra of 1 3C-enriched cellulose samples were the sum of 400 scans, collected using a standard C P pulse sequence, using a 3.0 ps proton 90° pulse, a 1000 ps contact pulse and a 2 s delay between repetitions for the cellulose from l ,3- ' 3 C-glycerol synthesis and a contact pulse of 2000 ps and 10 s delay between repetitions for the cellulose from the l 3C-4-glucose synthesis. l 5 N - C B M 2 a C P / M A S spectra were the sum of 12 000 scans, collected using a standard C P pulse sequence, using a 5.25 ps proton 90° pulse, a 2000 ps contact pulse and a 5 s delay between repetitions. For the CP-drain experiments, spectra were the sum of 31 232 scans, using 6.0 ps proton 90° pulse, a 1000 ps contact pulse, a 1 s delay between pulses with a 10 ms drain time. N o line broadening or resolution enhancement was applied during processing of the data for these C P experiments. 41 2.10.2 N M R in so lu t ion N M R spectra were recorded at 27°C on a Varian Unity 600 M H z spectrometer equipped with a gradient triple resonance probe. ' H chemical shifts were reference to an internal standard of DSS at 0.00 pp, and 1 5 N chemical shifts were referenced to external 2.9 M 1 5 N H 4 C 1 in 1 M HC1 at 24.93 ppm (71). This latter reference yields l 5 N chemical shifts 1.6 ppm greater than those obtained using liquid N H 3 (138). Spectra were processed an analyzed using F E L I X software (Biosym Technologies; San Diego, C A ) . 42 3. Results In the following section, I present data to show that C B M 2 a binds to the insoluble cellulose preparations P A S C , B M C C , and Avice l . The residues involved in the binding of C B M 2 a to B M C C were investigated by site-directed mutation. Solvent-exposed tryptophans are shown to be essential determinants of binding affinity and, with one exception, the other residues that are prominent on the tryptophan-rich binding face are not essential determinants of binding affinity. Additionally, each tryptophan plays a different role in binding. C B M 2 a is shown to bind specifically to the crystalline regions of cellulose; it shares sites with other binding modules that bind to crystalline cellulose ( C e l 6 A - C B M 2 a and C B M 3 ) and does not share sites with binding modules with specificity for cello-oligosaccharides and amorphous cellulose ( C B M 17 and C B M 4 - 1 ) or the reducing ends of cellulose chains (CBM9-2) . C B M 17 and C B M 4 - 1 compete for some sites on amorphous cellulose (PASC) , but there are sites that are uniquely recognized by each module. The binding of C B M 2 a was investigated in more detail. The rate of adsorption of C B M 2 a to P A S C is much slower than to B M C C and appears to be limited by mass transfer. Binding of C B M 2 a is apparently irreversible; the desorption isotherm is not equivalent to the adsorption isotherm, yet at equilibrium, C B M 2 a bound to the surface of B M C C exchanges with C B M 2 a in the solution phase, a rapid process where the binding equilibrium is re-established in less than 30 seconds. In preliminary experiments of the samples and technique, it was found that without further refinement, specific interactions between 1 5 N - C B M 2 a and 1 3C-enriched cellulose could not be detected by solid state N M R . 3.1 Production of His6-CBM2a H i s 6 - C B M 2 a was produced by E. coli using the expression vector p T u g K H 6 - I E G R - C B M 2 a . Most of it leaked from the periplasm into the culture supernatant. It was purified from the 4? supernatant by I M A C to >95% homogeneity, as estimated by Coomassie blue-stained S D S - P A G E gels (Figure 3.1). Yields routinely exceeded 100 mg of purified product per litre of culture. „ „ 1 2 3 4 5 6 7 8 9 ()- 4 66.2 45.0 * 31.0 * 21.5 f 6 5 / Figure 3.1 Purification of H i s 6 - C B M 2 a from concentrated E. coli culture supernatant. Lane 1, M W standards (size indicated in kDa); lane 2, concentrated supernatant loaded onto the charged I M A C column; lane3, column eluent; lanes 4, 5, 6, 7, and 8 are fractions eluted in I M A C binding buffer with step-wise increases of imidizole;30 m M , 50mM, 100 m M , 250 m M , and 500 m M respectively. Lane 9 is the fraction eluted with 100 m M E D T A . 3.2 Binding of CBM2a to Cellulose Crystallinities, relative to a highly crystalline preparation from Valonia ventricosa, for the cellulose preparations used in this work are 0.76 for B M C C (68) and 0.50 for Avicel (139) as measured by X-ray diffraction techniques. The crystallinity index for P A S C is not known, but is likely to be significantly less than that of Valonia due to the acid-swelling process used in its preparation. C C P / M A S spectra of these celluloses (Figure 3.2) also confirm some of these structural features. The peaks that correspond to C-4 (81 - 93 ppm) are indicative of cellulose structure (2, 4, 32). C-4 atoms that are part of the internal, highly ordered crystalline structure give rise to a sharp downfield peak at approximately 89 ppm; C-4 atoms at the cellulose surface or part of amorphous structures (4), are less highly ordered and 44 produce the broader, less intense, upfield peak at 84 ppm. Based on relative areas of these two C-4 peaks, B M C C has a large proportion of internal C-4 atoms and P A S C has a larger proportion of C-4 exposed to the solvent. The spectrum of Avice l appears to be a mixture of the other two spectra. Paradoxically, the bulk crystallinity of P A S C appears very low, as evidenced by the predominant broad C-4 peak and the much less discernible downfield sharp peak from the crystalline bulk, yet C B M 2 a binds to P A S C with high capacity. The affinity of H i s 6 - C B M 2 a for these insoluble cellulose preparations decreased in the order B M C C > P A S C > A v i c e l (Table 3.1; Figure 3.3) and ranged from 3.2 x 106 M ' 1 to 1.0 x 106 M " 1 , suggesting preferential binding of C B M 2 a to crystalline cellulose. It should be noted that the affinities reported here are average values based on the mass of each cellulose sample, not the available surface area. Furthermore, it is assumed that binding can be validly described by a Langmuir-type binding expression. The binding properties of the wild-type C B M 2 a , produced by proteolytic cleavage and separation of the binding module from the intact enzyme (Xyn lOA) , on these substrates were indistinguishable from the H i s 6 - C B M 2 a construct (data not shown). Hereafter, C B M 2 a indicates either C B M 2 a or H i s 6 - C B M 2 a because the molecules are functionally indistinguishable. Since the hexahistidine tag does not affect binding, it was not removed from any of the wild-type and mutant polypeptide constructs. Table 3.1 Binding affinity and capacity for the adsorption of C B M 2 a to insoluble cellulose at 4°C in 50 m M potassium phosphate, p H 7.0. Insoluble Affinity (Ka) Capacity ([N0]) AG cellulose (106 M"1) (umolg"1 cellulose) (kJ/mol) B M C C 3.2 ± 0 . 3 1 1 . 7 ± 0 . 3 -34.510.2 P A S C 1.510.1 14.310.4 -32.810.2 Avice l 1.0 ± 0 . 1 2.410.1 -31.810.3 45 Based on isothermal titration calorimetry (ITC) data, highly crystalline B M C C preparations offer at least two classes of binding sites to C B M 2 a , both of relatively high affinity and characterized by binding constants (K a ) within the range reported here (10 6-10 7 M" 1) (25). Thus, possibly due to differences in the surface energies of its various crystal faces, crystalline cellulose appears to present a heterogeneous array of binding sites to C B M 2 a . The K a values reported in table 3.1, however, assume that the cellulose surface is uniform and offers only a single class of binding site. This approach is justified by the fact that the shape of the cumulative isotherm generated by the depletion method (used here) is relatively insensitive to the energetics and occupancy of lower affinity/lower occupancy sites (54, 70) as opposed to that of the differential binding isotherm, provided by ITC. The K , regressed from the cumulative depletion isotherm using the Langmuir equation (Equation 1, section 2.7) is therefore an averaged value which reflects, in part, the relative contributions of each class of binding site on the cellulose surface. Based on this mode of analysis, the lower K a values reported for binding of C B M 2 a to Avicel and P A S C , compared with the affinity for B M C C , suggest that these forms of insoluble cellulose present a higher fraction of low affinity sites to C B M 2 a resulting in a lower K a calculated from the cumulative isotherm. The nature of these low-affinity sites, however, is unclear. They could reflect natural differences in the abundance of high and low energy cellulose crystal faces. They could also originate from the ability of C B M 2 a to bind both crystalline and amorphous morphologies of cellulose, with the affinity of the module for amorphous regions being somewhat weaker. The latter possibility was tested by competition isotherms measuring the binding of C B M 2 a to P A S C in the presence and absence of C B M s that recognize only the amorphous regions of P A S C , namely C B M 4 - 1 and C B M 17 (; section 3.4.6). 47 | C-2,3,5 (A) Avicel • C 4 • C-6 i 1 (B) BMCC 110 100 90 80 70 60 50 ppm Figure 3.2 C P / M A S Spectra of insoluble cellulose: (A) Avicel , (B) B M C C , and (C) P A S C . The horizontal bars above the spectrum of Avicel indicate the spectral ranges of the corresponding carbon atoms of the glucose monomer unit of cellulose and apply to B M C C and P A S C as well 48 0 1 2 3 4 5 6 7 8 9 10 Free CBM2a (u.M) Figure 3.3 Binding of C B M 2 a to insoluble cellulose P A S C (O) , B M C C ( • ) and Av ice l (A). Binding data (Table 3.1) is regressed using the Langmuir binding expression (Equation 1) 49 3.3 Binding of CBM2a mutants to BMCC Using the synthetic CBM2a-encoding D N A (pTugK-H6-LEGR-CBM2a) as a template and appropriate primers (Table 2.2), fourteen mutants of C B M 2 a were constructed by two-primer P C R cassette mutagenesis (see Section 2.4), including conservative (phenylalanine and tyrosine) and non-conservative (alanine) mutants of each of the three tryptophan residues, W17, W54, and W72, on the binding face. Other residues on the binding face of C B M 2 a that have the potential to participate in hydrogen bonds with the cellulose surface were substituted individually with alanine (Figure 3.4). Since C B M 2 a binds well to B M C C , it was chosen as the matrix for comparison of the affinities of the wild-type and mutant C B M 2 a polypeptides. In a later section, it wi l l be shown that C B M 2 a localizes to crystalline regions of cellulose. 3.3.1 Tryptophan mutants Substitution of each of the surface tryptophan residues with tyrosine, phenylalanine or alanine reduced the affinity for B M C C , with alanine having a greater effect than the more conservative phenylalanine substitution (Table 3.2). The affinities of the tyrosine mutants were W72Y>W17Y>W54Y; those of the phenylalanine mutants were W72F>W17F>W54F; and those of the alanine mutants were W17A>W72A>W54A. The W 5 4 A mutant had the lowest affinity, two orders of magnitude lower than the wild-type, whereas the W 7 2 Y mutant had the highest affinity of the nine tryptophan mutants, about two-thirds of the wi ld type (Table 3.2). Most of the binding energy (AG) is provided by the tryptophan residues. The sum of the A A G values (Table 3.2) of the tryptophan to alanine mutants is -21.2 kJ/mol and accountss for approximately 61% of the total binding energy of the wild-type binding module. 50 Table 3.2 Binding affinity of C B M 2 a tryptophan variants for B M C C at 4°C in 50 m M potassium phosphate, p H 7.0. CBM2a Variant K a (10 6 M-1) AG (kJ/mol) AAG (kJ/mol) H i s 6 - C B M 2 a 3.2 ± 0.3 -34.5 ± 0.2 W17F 0.6 ± 0.1 -30.8 ± 0.2 -3.7 ± 0 . 4 W 1 7 Y 1.6 ± 0.2 -32.3 ± 0 . 3 -2.2 ± 0 . 5 W 1 7 A 0.5 ± 0.1 -30.1 ± 0 . 6 -4.1 ± 0 . 8 W54F 0.36 ± 0.03 -29.5 ± 0 . 2 -5.0 ± 0 . 4 W 5 4 Y 0.34 ± 0.01 -28.7 ± 0 . 1 -5.8 ± 0 . 3 W 5 4 A 0.03 ± 0.02 -24.0 ± 3 . 0 -11.0 + 3.0 W72F 1.7 ± 0.1 -33.1 ± 0 . 2 -1.4 ± 0 . 4 W 7 2 Y 2.3 ± 0.2 -33.7 ± 0 . 3 -0.8 + 0.5 W 7 2 A 0.21 ± 0.01 -28.2 ± 0 . 1 -6.3 ± 0 . 3 3.3.2 O t h e r m u t a n t s Alanine was substituted for other residues on the binding face that could potentially form hydrogen bonds with cellulose. The mutation N 8 7 A reduced binding to half that of the native polypeptide; the N 1 5 A mutation reduced binding to one-fifth of native affinity. The mutations, N18 and Q52A reduced binding only slightly; the mutation Q83A did not affect binding (Table 3.3). The sum of the A A G values of these mutants is -6.6 kJ/mol and, when added to the contribution made by the tryptophan to alanine mutants, is -27.8 kJ/mol, accounts for 80% of the total binding energy. There are a few other residues on this binding face that contribute to the binding of C B M 2 a to cellulose; these could be discovered by continued site-directed mutation studies. Table 3.3 Binding affinity of C B M 2 a variants for B M C C at 4°C in 50 m M potassium CBM2a Variant K a (10 6 IVT1) AG (kJ/mol) AG (kJ/mol) H i s 6 - C B M 2 a 3.2 ± 0.3 -34.5 ± 0 . 2 N15A 0.47 ± 0.02 -30.1 ± 0 . 2 -4.4 ± 0.4 N 1 8 A 2.9 ± 0.2 -34.3 ± 0 . 2 -0.2 ± 0.4 Q52A 2.7 ± 0.2 -34.1 ± 0 . 2 -0.3 ± 0.4 Q83A 3.4 ± 0.3 -34.6 ± 0 . 2 0.1 ± 0 . 4 N 8 7 A 1.6± 0.1 -32.8 ± 0 . 2 -1.7 ± 0 . 4 Figure 3.4 Two views of C B M 2 a . The mutated residues are rendered using balls and sticks and are highlighted; surface tryptophans are green and the other polar residues are red. This figure was created with M o l M o l (63) based on the N M R solution structure (141). 52 3.4 Competition Binding Isotherms The binding specificities of many C B M s are known. Types B and C bind to soluble ligands; type A C B M s bind to insoluble cellulose and to P A S C , but it is not precisely known i f this type also binds to amorphous cellulose and soluble oligosaccharides. Using the complex substrate P A S C , that binds all types of C B M (Table 3.4) and has crystalline and amorphous regions, binding competition experiments, involving two C B M s at a time, were performed to determine whether C B M s from the three types compete for binding sites. 3.4.1 C B M s b ind to P A S C a n d B M C C C B M s from each of the three types were used in competition experiments. Type A C B M s were C B M 2 a , C e l 6 A - C B M 2 a and C B M 3 ; type B C B M s were C B M 4 - 1 and C B M 17; type C was C B M 9 - 2 . A l l of the C B M s used in the competition binding experiments bound to P A S C (Table 3.4); type A C B M s (CBM2a , C e l 6 A - C B M 2 a and C B M 3 ) also bind to B M C C (Table 3.4). Binding capacity of P A S C for the C B M s varied from 6.1 pmolg"' for C B M 4 - 1 to 24.0 pmolg"' for C B M 1 7 . It should be noted however that type A C B M s had a similar capacity for P A S C (-15 pmol-g"1) and a similar capacity for B M C C (-11 pmol-g"1). The C B M binding parameters reported in Table 3.4 were each determined by a depletion isotherm, as described in section 2.7. The concentration of each C B M was calculated from its A 2 8 0 (corrected for light scattering) and molar extinction coeffecient (Table 2.3). 53 Table 3.4 C B M binding affinity and capacity for B M C C and P A S C at 4°C in 50 m M potassium phosphate, p H 7.0. C B M P A S C B M C C Affinity (K a ) (10 6 M'1) Capacity ([N0]) ( umo lg 1 cellulose) Affinity (K a ) (10 6 M*1) Capacity ([No]) (umolg- 1 cellulose) C B M 2 a C e l 6 A - C B M 2 a C B M 3 C B M 4 - 1 C B M 17 C B M 9 - 2 1.5 (±0 .1 ) 0.9 (± 0.3) 1.1 (±0 .2 ) 0.25 (± 0.02) 1.1 (±0 .1 ) 0.63 (± 0.04) 14.3 (± 0.4) 14.9 (± 1.1) 17.4 (± 0.8) 6.1 (±0 .1 ) 24.0 (± 0.8) 8.4 (±0 .1 ) 3.2 (±0 .3 ) 3.3 (±0 .8 ) 1.0 (± 0.1) 11.7 (±0 .3 ) 10.3 (±0 .4 ) 12.4 (±0 .3 ) 3.4.2 Labell ing of C B M s Five to ten milligrams of each C B M used in the competition binding experiments were labelled with either A M C A - X or Oregon Green®. These probes react with primary amines, the N-terminus or lysine residues, of polypeptides. Each binding module therefore wil l have at least one potential labelling site (Table 3.5). The labelling efficiency of each reaction was calculated based on the extinction coefficients at their maximum absorption wavelength for each label (18 500 M ' - cm" 1 at 352 nm for A M C A - X and 86 200 M ' - c m " 1 at 506nm for Oregon Green®; Molecular Probes). These two fluorescent probes were chosen because they have well separated excitation and emission properties (Figure 3.5). Labelling efficiency ranged from approximately 0.42 to 1.21 moles of fluorescent probe per mole of polypeptide. In competition experiments, due to the sensitivity of the fluorimeter, each labelled C B M was diluted with unlabelled C B M so that maximum fluorescence measured (-1000 fluorescence units) would coincide with the maximum protein concentration used in a binding isotherm, typically 30 p M . In other words, labelled C B M was diluted in unlabelled C B M so that every C B M sample fell roughly into the same reading range of the fluorimeter. 54 Table 3 . 5 C B M s , labels and number of potential labelling sites for C B M s used in competition binding experiments. CBM Number of Label Extent of Labelling potential labelling sites (Molar ratio Label/CBM) C B M 2 a 2 Oregon Green 0.42 C B M 2 a 2 A M C A - X 1.21 C B M 4 - 1 1 A M C A - X 0.60 C e l 6 A - C B M 2 a 2 A M C A - X 0.65 C B M 17 9 Oregon Green 0.65 C B M 3 11 not labelled -C B M 9 - 2 * 16 A M C A - X 0.78 *CBM9-2 labelled with AMCA-X did not bind to cellulose 3.4.3 S t a n d a r d s a n d Isotherms by f l u o r e s c e n c e A standard or calibration curve for each of the labelled C B M preparations and each cellulose substrate used in the competition experiments was created so that the concentration of each C B M could be measured by fluorescence. To generate each curve, a binding experiment was performed for each labelled C B M and cellulose substrate, using untreated microcentrifuge tubes (ie. without preadsorption with B S A ) . A control sample containing C B M but no cellulose was also prepared in an identical microcentrifuge tubes. The fluorescence and net A 2 8 0 of each sample, including the control, was measured; the molar concentration protein in the solution phase of each sample was then used to construct calibration curves for both cellulose-containing and cellulose-free samples ( P A S C , Figure 3.6; B M C C , Figure 3.7). There was a linear relationship between fluorescence and the solution concentration of free or total C B M samples. Depletion-type isotherms were then constructed from fluorescence data. The free-protein concentration was determined by using the calibration curve constructed in the presence of cellulose; total-protein concentration was determined by using the calibration curve constructed in the absence of cellulose. Each of the two callibration curves were linear, but the calibration curve for free-protein did not fall on the same line as 55 the total-protein standard. Unless otherwise specified, a C B M preparation (eg. C B M 2 a -OG) contains a mixture of labelled and unlabelled C B M . In previous work, it was shown that the binding of C B M 2 a to insoluble cellulose is not affected by the presence of F ITC (57). Similarly, the Oregon Green and A M C A - X labels should not perturb binding of C B M 2 a . This was verified by determining that the Langmuir binding constants for labelled and unlabelled C B M 2 a preparations, where the concentration was calculated by U V absorbance (net A 2 8 0 ) , in binding to P A S C were essentially equivalent (Fig 3.8-A; Table 3.6). The effect of competition between the specific binding of C B M 2 a to cellulose and the non-specific binding of protein to cellulose was assessed by determining the binding constant of C B M 2 a in the presence of approximately 20 u\M B S A . The binding of C B M 2 a was not affected (see section 3.4.4; Table 3.6). It was observed that C B M 2 a bound, non-specifically, to the sides of the microcentrifuge tubes. To minimize the effect of non-specific adsorption of C B M s affecting competition results, B S A was used to block the tubes used in competition experiments. The B S A pre-treatment did not affect the C B M ; binding parameters of C B M 2 a measured by fluorescence (Table 3.6) using treated tubes was essentially equivalent to those calculated for C B M 2 a isotherms performed in untreated tubes, measured by U V absorbance. For the other C B M s used in the competition experiments (Table 3.5) the residues on each of the polypeptides that may potentially react with the fluorescent probes appeared well removed from the putative ligand binding sites highlighted in figures 1.4, 1.5 and 1.6. In experiments similar to the one described above, the binding of C B M 2 a , C e l 6 A - C B M 2 a , C B M 4 - 1 , or C B M 17 to cellulose was not affected by the presence of a fluorescent probe (data not shown). The labelling of C B M 9 - 2 with A M C A - X blocked its binding to P A S C . Competition experiments involving C B M 9 - 2 were therefore performed using unlabelled 56 polypeptide. Because of solubility problems with the protein preparation, unlabelled C B M 3 was also used unlabelled. 3.4.4 CBM2a adsorption to PASC in the presence of BSA Microcentrifuge tubes used in competition experiments were treated with B S A to minimize the amount of non-specific binding of C B M s to the walls of tubes. The binding of C B M 2 a -O G was not affected by the presence of B S A (Figure 3.8-B; Table 3.6). The association affinity constants of C B M 2 a alone and in the presence of 20 p M B S A , as measured by fluorescence were similar and within the standard error of regression. Additionally, the 20 pmol-g 1 capacity ([N 0]) of P A S C to bind C B M 2 a - O G was not affected by the presence of B S A . Since the non-specific adsorption of B S A to the microcentrifuge tubes, or the cellulose, did not affect the specific adsorption of C B M 2 a to cellulose, the B S A was used to block the microcentrifuge tubes in the subsequent competition experiments. Table 3.6 Binding of C B M 2 a to P A S C : U V absorbance, fluorescence and B S A competition at 4°C in 50 m M potassium phosphate, pH 7.0. CBM2a Affinity Capacity (Ka: 106 IVT1) ([N0]: umolg"1) U V Absorbance (unlabelled) 1.5 (± 0.1) 14.3 ( ± 0 . 4 ) * U V Absorbance (CBM2a-OG) 1.6 (± 1.0) 22.0 (± 3.0) Fluorescence 2.3 (± 0.4) 20.0 (±1 .0 ) Fluorescence with 20 p M B S A 2.2 (± 0.4) 20.8 (± 0.9) *The capacity measured for CBM2a unlabelled was for a preparation of PASC different from that used for the CBM2a-OG experiments listed. PASC preparations differ in total available surface area, therefore only [N0] is affected. 57 350 375 400 425 450 475 500 525 550 575 600 Emission wavelength (nm) Figure 3.5 Emission spectra of C B M 2 a labelled with Oregon Green or A M C A - X . C B M 2 a -O G was excited at 506 nm, emission was measured from 510 nm to 600 nm using a 530 nm emission filter; C B M 2 a - A X was excited at 352 nm, emission was measured from 360 nm to 600 nm. For comparison of the two labels, the fluorescence emission was normalized to the maximum fluorescence value measured for each label. 58 Figure 3.6 Standard curves of fluorescence vs protein concentration (uM) for each of the preparations of C B M s used in the P A S C binding competition experiments (clockwise from top left): C B M 2 a - O G , C B M 2 a - A X , C B M 4 - 1 - A X and C B M 1 7 - O G . For each C B M , two calibration curves were plotted: fluorescence vs. C B M concentration for the total protein samples (incubated without celluose) (O),: fluorescence vs. C B M concentration for the free protein samples (incubated with cellulose) ( • ) . Free C B M concentration was calculated using the calibration curve constructed using free protein samples; total C B M concentration was calculated using the calibration curve constructed using total protein samples. 59 Figure 3 . 7 Standard curves of fluorescence vs. protein concentration (pM) for C B M 2 a - O G (top left), and C B M 2 a - A X ( t o p right) and Ce l6A-C B M 2 a - A X (lower right) preparations used in the B M C C binding competition experiments. Standards of free protein ( • ) and total polypeptide (O) were considered separately. [Cel6A-CBM2a-AX] (uM) 60 Figure 3.8 Binding of C B M 2 a - O G to P A S C . (A) C B M 2 a - O G adsorption to P A S C measured by U V absorbance ( • ) and by fluorescence (O) . (B) C B M 2 a - O G adsorption to P A S C alone ( • ) and in the presence of 20 p M B S A 61 3.4.5 A m o d e l of compet i t ion b ind ing Knowing the binding parameters of two binding modules competing for a single class of binding sites on a sorbant surface, the extent of competition can be predicted using derivatives of the Langmuir-like binding model. When two binding modules compete for the same binding site, it is the apparent affinity of each that is affected. For example, in an hypothetical competition binding experiment involving two C B M s , called C B M - 1 and C B M -2, have an affinity, K a l and K a 2 , respectively, for the same binding site (capacity [N 0]), then the apparent K a l ( K a l a p p a r e n t ) would be lower in the presence of C B M - 2 than the K a l measured in the absence of C B M - 2 . The capacity ([N 0]) is the sum of the concentration of unoccupied binding sites ([N]) and the concentration of sites occupied by C B M - 1 is [B,] the concentration of sites occupied C B M - 2 is [B 2 ] . [N0] = [N] + [Bl] + [B2] (3) The affinity of C B M - 1 ( K a l ) is described by: K = [ B i ] ( 4 ) al [Ft][N] substituting equation 3 into equation 4, gives: K W W ^ _ r A l \ _ . (6) [B2i [B,] = 0 + .^^ ]) By definition, [B,] = [No]K a l A p p a r e n l [F , ] (7) (l + K a l [F,]) At low concentrations of C B M - 1 (i.e. [F,]->0) K = v a\Apparent a\ \ \B^ (8) [ 2] 62 In the presence of a large concentration of C B M - 2 , C B M - 1 wi l l appear to bind with a lower apparent affinity ( K a l A p p a r e n t ) . Similarly, if the K a A p p a r e n l of a C B M is approximately equal to its K a in the absence of a competitor, then the two C B M s do not compete for the same binding sites. Thus, changes in K a are a sensitive indicator of binding competition. A n effective plot to aid interpretation of binding competition is the fraction of saturation ([B 2]/[N 0]) of a C B M vs. the fraction of saturation of the second C B M ([B,]/[N 0]). A t a [B,]/[N 0] equal to 1, the surface is fully saturated with C B M - 1 ; at [B,]/[N 0] equal to 0, no C B M - 1 is bound. If two C B M s bind to separate sites, and do not compete, this plot is a horizontal line, with a slope of zero; C B M - 2 bound to the surface is not affected by the binding of C B M - 1 and, therefore, the C B M s are recognizing different sites. In the case where C B M - 1 and C B M - 2 compete for exactly the same sites, then the plot has a negative slope, with a x-intercept of 1; as the concentration of C B M - 1 approaches saturation, C B M - 2 is displaced and the fraction of C B M - 2 bound approaches zero. The actual slope of the [B 2]/[N 0] vs. [B,]/[N 0] plot depends on the starting concentration of the competitor ( C B M - 2 in this example) and the percentage of binding sites common to both C B M s . To validate of this model of competition, two binding experiments were performed involving the competition between two labelled C B M 2 a preparations ( C B M 2 a - A X and C B M 2 a - O G ) on B M C C (Figures 3.9 and 3.10). In this case, the fraction of saturation of C B M 2 a - A X ([B]/[N 0]) vs. the fraction of saturation of C B M 2 a - O G ([B]/[N 0]) is plotted (Figures 3.9-B and 3.10-B); the competitor ( C B M 2 a - A X at constant concentration of either 11 p M or 15 p M ) is plotted on the Y-axis. In both cases, where the competitor C B M 2 a - A X concentration was held constant at either 11 p M (Figure 3.9) or 15 p M (Figure 3.10), as C B M 2 a - O G approached saturation ( C B M 2 a - O G [B]/[N 0] approaches 1), the fraction of C B M 2 a - A X 63 bound to the surface of B M C C ( C B M 2 a - A X [B]/[N 0]) approached zero, as would be expected. At high concentrations of C B M 2 a - O G ( [B C B M 2 a . O G ] / [N 0 ] -0.5) the data depart slightly from the line predicted by ideal competition conditions (shown by a dashed line in Figures 3.9-B and 3.10-B). At these concentrations of C B M 2 a - O G , the total concentration of C B M 2 a ( C B M 2 a - A X plus C B M 2 a - O G ) is virtually at saturation (>40 p M ) and secondary binding effects, such as protein-protein layering, are likely occurring and could account for the deviation from the predicted competition. Additionally, the capacity of C B M 2 a - O G , when in competition with C B M 2 a - A X , is expected to eventually reach the [N 0] of C B M 2 a - O G that is observed without competition (Figures 3.9-A and 3.10-A); this is not seen at the range of C B M 2 a - O G concentrations tested when in competition. The apparent affinity of C B M 2 a - O G can be predicted using this theory. At 11 p M , bound C B M 2 a - A X is at -82% saturation; at 15 p M , the B M C C substrate is - 95% saturated. The apparent affinity of C B M 2 a - O G in competition with C B M 2 a - A X , predicted by the fraction of saturation (equation 8), is 0.78 x 10 6 M _ 1 and 0.22 x 106 M 1 , respectively, and agrees remarkably well with the observed values (Table 3.7). Table 3.7 C B M 2 a - O G in competition with C B M 2 a - A X on B M C C at 4°C in 50 m M C B M * Compet i tor C B M 2 a -OG Figure K a apparent (106 M"1) C B M 2 a - O G 3 - 4.3 (±0 .5 ) 3.9-A C B M 2 a - O G 3 C B M 2 a - A X (11 p M ) 0.72 (±0 .05) 3.9-A C B M 2 a - O G 3 C B M 2 a - A X ( 1 5 p M ) 0.45 (± 0.03) 3.10-A 64 Figure 3.9 Binding of C B M 2 a - 0 G to B M C C in the presence of C B M 2 a - A X . A ) Binding of C B M 2 a - O G alone ( • ) and in the presence of 11 p M C B M 2 a - A X (O) ; C B M 2 a - A X bound vs. C B M 2 a - O G free is also plotted (A). B) Fraction bound C B M 2 a - A X ([B]/[N„]) vs. fraction bound C B M 2 a - O G ([B]/[N ( 1]); solid line is the linear regression of the data, the dashed line is the theoretical regression assuming C B M 2 a - A X saturation of 0.82 and ideal competition between C B M 2 a - A X and C B M 2 a - O G 65 Figure 3.10 Binding of C B M 2 a - O G to B M C C in the presence of C B M 2 a - A X . A ) Binding of C B M 2 a - O G alone ( • ) and in the presence of 15 u M C B M 2 a - A X (O) ; C B M 2 a - A X bound vs. C B M 2 a - O G free is also plotted ( • ) . B) Fraction bound C B M 2 a - A X ([B]/[NJ) vs. fraction bound C B M 2 a - O G ([B]/[N ( )]); solid line is the linear regression of the data, the dashed line is the theoretical regression assuming C B M 2 a - A X saturation of 0.95 and ideal competition between C B M 2 a - A X and C B M 2 a - O G 66 3.4.6 C B M 2 a in compet i t ion with C B M 4 - 1 , C B M 1 7 a n d C B M 9 - 2 C B M 4 - 1 binds specifically to amorphous cellulose, showing no significant affinity for crystalline preparations of cellulose such as V. ventricosa cellulose and B M C C (23, 64, 125). The K a for C B M 4 - 1 binding to either soluble cellulose polymers or P A S C at 4°C (50 m M potassium phosphate buffer, pH 7.0) is 2.5 x 105 M - ' (Table 3.4), an order of magnitude less than the average K a for C B M 2 a binding to P A S C . C B M 17 has specificity that is similar to C B M 4 - 1 ; it binds cello-oligosaccharides or amorphous cellulose with a maximum affinity for P A S C of 7.6 x 105 M 1 (Table 3.4). Competition experiments should determine whether the family 2a and the family 4 and 17 C B M s share the same sites on cellulose. Thus, in a competition experiment at fixed total concentration of C B M 4 - 1 , increasing concentrations of C B M 2 a should lead to the progressive exclusion of bound C B M 4 - 1 if C B M 2 a shows an affinity for amorphous regions of P A S C . Similarly, for a fixed total C B M 2 a concentration, increasing concentrations of C B M 4 - 1 or C B M 17 should lead to a progressive displacement of C B M 2 a . Binding of C B M 2 a to P A S C was not significantly affected by the presence of C B M 4 - 1 (Figure 3.11; Table 3.8). Alone, C B M 2 a binds to P A S C with a K a of 1.2 (± 0.1) x 106 M 1 . When C B M 4 - 1 is present at a total concentration of 19 p M (an amount that is approximately 95% of C B M 4 - 1 saturation), the apparent K a for binding of C B M 2 a to P A S C is reduced only slightly to 1.0 (± 0.1) x 106 M " ' ; in the presence of 14.8 p M C B M 4 - 1 , approximately 80% of saturation, the apparent K a of C B M 2 a was again only slightly lower than its corresponding C B M 4 - 1 free value (Table 3.8). The amount of bound C B M 4 - 1 decreased no more than 10% (Figure 3.11-C) as C B M 2 a approached saturation, indicating that the vast majority of bound CBM4-1 is unaffected by the presence of C B M 2 a . 67 The binding of neither C B M 4 - 1 nor C B M 17 was affected by the presence of C B M 2 a . The affinity of C B M 4 - 1 was 0.16 (± 0.02) x 10 6 M ' 1 and did not change in the presence of C B M 2 a (Figure 3.12; Table 3.8). As C B M 4 - 1 approached saturation, only a very small population of bound C B M 2 a (<5%) was displaced by the binding of C B M 4 - 1 ([Figure 3.12-B). The affinity of C B M 1 7 decreased insignificantly from 0.76 (± 0.08) x 105 M " ' to 0.67 (± 0.04) x 105 M " 1 in the presence of C B M 2 a at approximately 85% of saturation ([Figure 3.13; Table 3.8). A t a high concentration of C B M 1 7 (near saturation) only about 10% of the bound C B M 2 a was displaced by the binding of C B M 1 7 (Figure 3.13-B). These results suggest that C B M 2 a occupies separate sites distinct from those occupied by C B M 4 - 1 and C B M 17, with a possible 5-10% overlap. C B M 9 - 2 binds specifically to the reducing ends of sugars (A. B . Boraston et al,; V . Notenboom et al., in press); its binding site accommodates only two sugar rings, such as cellobiose. It binds to P A S C with an affinity of 0.63 (± 0.04) x 106 M " ' at a sorbant capacity of 8.4 (±0 .1 ) j imol-g 1 . The binding of C B M 2 a was not affected by the presence of C B M 9 - 2 (Figure 3.14; Table 3.8) at a lower saturation; in the presence of more C B M 9 - 2 , both the affinity and binding capacity of C B M 2 a - O G decreased only slightly (Figure 3.14; Table 3.8) Together, these results suggest that C B M 2 a binding is specific to crystalline regions of P A S C . C B M 2 a does not share binding sites with binding modules with specificity for cello-oligosaccharides or amorphous cellulose (CBM4-1 and C B M 1 7 ) ; it also does not share binding sites with C B M 9 - 2 , which binds to the reducing ends of cellulose polymers. The small amount of competition between bound C B M 2 a and these other C B M s presumably occurs only at boundaries between crystalline and amorphous microstructures or reducing ends of cellulose molecules. 68 Table 3.8 P A S C competition binding summary I: C B M 2 a , C B M 4 - 1 , C B M 17 and C B M 9 -2 at 4°C in 50 m M potassium phosphate, p H 7.0. C B M Compet i tor K a Apparent of C B M Figure (106 M' 1) C B M 2 a - O G (none) 1.2 (±0 .1 ) 3.11 C B M 2 a - O G C B M 4 - 1 - A X (19 p M ) 1.0 (±0 .1 ) 3.11-A C B M 2 a - O G C B M 4 - 1 - A X (14.8 p M ) 0.96 (± 0.04) 3.11-B C B M 4 - 1 - A X " ( n o n e ) 0 . 1 6 ( ± 0 7 0 2 ) " 3 . 1 2 C B M 4 - 1 - A X C B M 2 a - O G ( 1 8 j i M ) 0.16 (±0 .02) 3.12 C B M 1 7 - O G ( n o n e ) 0 . 7 6 ( " ± 0 . 0 8 ) " 3 J"3 C B M 1 7 - O G C B M 2 a - A X (18.8 p M ) 0.67 (± 0.04) 3.13 C B M 2 a - O G C B M 9 - 2 (8 p M ) 0.98 (± 0.09) 3.14 C B M " 2 " a - " 6 G " * " ( " n o n e " ) 2 . 3 " ( ± 0 . 4 ) 3 T 4 - A " " " C B M 2 a - O G * C B M 9 - 2 (18.6 p M ) 1.7 (±0 .2 ) 3.14-B *a second preparation of C B M 2 a - O G 69 o.oo f 0 1 2 3 4 5 6 7 8 9 10 11 Free CBM2a-OG (uM) G) Figure 3.11 Binding of C B M 2 a - O G to P A S C in the presence of C B M 4 - 1 . (A) C B M 2 a - O G adsorption to P A S C alone ( • ) and in the presence of 95% saturating concentration of C B M 4 - 1 (O) where the fraction of C B M 4 - 1 -A X bound vs. C B M 2 a - O G Free (A) is also plotted. (B) C B M 2 a - O G adsorption to P A S C alone ( • ) and in the presence of 80% saturating concentration of C B M 4 - 1 (O) where the fraction of C B M 4 - 1 - A X bound vs. C B M 2 a - O G Free (A) is also plotted. (C) Fraction C B M 4 - 1 bound ([B]/[N ( )]) vs. Fraction C B M 2 a - O G bound for both saturation concentrations of C B M 4 - 1 - A X : 95% ( • ) , and 80% (Q). 1.00H £ 0.50-0.25-0.001 I 0.00 0.25 0.50 0.75 1.00 B/[N„] CBM2a B) 70 A ) 1.00-0.75 £ • ° - 5 0 0.25 0.00 CBM4-1 CBM4-1 w i t h C B M 2 a C B M 2 a 6 8 10 12 14 Free C B M 4 - 1 (uM) 16 18 20 22 B) 1.00 0.75 CQ O CQ 0.50 0.25 0.00 0.00 0.25 0.50 0.75 1.00 [B] / [N 0 ] C B M 4 - 1 Figure 3.12 Binding of C B M 4 - 1 - A X to P A S C in the presence of C B M 2 a - O G . A ) C B M 4 - 1 -A X adsorption to P A S C alone ( • ) and in the presence of C B M 2 a - O G (O) at 65% of saturation concentration. B) Fraction C B M 2 a - O G bound vs. fraction C B M 4 - 1 - A X bound 71 l.(X> A ) (I.5IIH • C B M 1 7 alone o C B M 1 7 with 18.8 uM C B M 2 a - A X A C B M 2 a - A X Bound Free CBM17-OG (\lM) 1.00 B) 0.75H ea u 0.50' 0.25H 0.00H 0.00 0.25 0.50 [B]/[N0 ]CBM17 0.75 1.00 Figure 3.13 Binding of C B M 1 7 - O G to in the presence of C B M 2 a . C B M 17 binding to P A S C alone ( • ) and in the presence of C B M 2 a (O) at approximately 85% of saturation; C B M 2 a -A X bound vs. C B M 1 7 - O G Free(A) is also plotted. B) Fraction C B M 2 a - A X bound ([B]/[N„]) vs. Fraction C B M 1 7 - O G bound. 72 A) 0 1 2 3 4 5 6 7 8 9 10 11 Free CBM2a-OG (u.M) Figure 3.14 Binding of C B M 2 a - O G in the presence of C B M 9 - 2 . A ) C B M 2 a binding to P A S C alone ( • ) and in the presence of 8 «M C B M 9 - 2 (O). B ) C B M 2 a binding to P A S C alone and in the presence of 18.6 «M C B M 9 - 2 . These two competition experiments were done using two separate preparations of C B M 2 a - O G , hence the slight differences in affinity of C B M 2 a - O G alone. 73 3.4.7 C B M 2 a in compet i t ion with C e l 6 A - C B M 2 a a n d C B M 3 The binding module from C. cellulovorans cellulose-binding protein A ( C c C b p A - C B M 3 , hereafter C B M 3 ) , binds to insoluble cellulose (40, 41). A crystal structure has been solved for the related C. thermocellum scaffoldin subunit C t C i p C - C B M 3 (130); the C B M 3 amino acid sequence is very similar to that of C t C i p C - C B M 3 (130), has similar binding characteristics, and presumably a similar structure. C t C i p C - C B M 3 has an array of exposed aromatic residues characteristic of C B M s that bind to crystalline cellulose. The affinity of C B M 3 for P A S C and B M C C is very similar to that of C B M 2 a (~106 M 1 ) . With C B M 3 present as a competitor (not labelled) at a constant total concentration of 15 p M (approximately 72% of saturation) the apparent affinity of C B M 2 a - O G for P A S C is significantly decreased. This demonstrates that there are sites shared by both C B M 3 and C B M 2 a (Figure 3.15; Table 3.9). In this experiment, the affinity of C B M 2 a - O G was slightly lower than that measured in other experiments likely due to incomplete separation of free Oregon Green from the C B M 2 a - O G conjugates in the preparation of the labelled C B M 2 a -O G . However, the free fluorescent probe would not account for the apparent reduction in affinity of C B M 2 a observed in the presence of C B M 3 . C e l 6 A - C B M 2 a (previously referred to as C B D C e n A ) and X y n l O A - C B M 2 a (CBM2a) share similar binding properties and apparent binding specificity for cellulose with a significant degree of crystallinity (27, 37). X y n l O A - C B M 2 a and C e l 6 A - C B M 2 a share 52% amino acid sequence identity with 67% aa sequence similarity, including the three surface tryptophan residues implicated in the binding of family 2a C B M s to insoluble cellulose (12, 27, 94). These two family 2a binding modules bind similarly to the insoluble cellulose preparations P A S C and B M C C (Table 3.1 and Table 3.4) with an affinity for P A S C of approximately 1 x 10 6 M _ 1 and an affinity for B M C C of approximately 3 x 106 M " 1 . In competition experiments 74 using a constant amount of C e l 6 A - C B M 2 a - A X as a competitor, the apparent affinity of C B M 2 a - O G for both P A S C (Figure 3.16) and B M C C (3.17) was significantly reduced (Table 3.9). A majority of C e l 6 A - C B M 2 a bound to the cellulose surfaces directly competes with C B M 2 a (Figure 3.16-B and Figure 3.17-B); as C B M 2 a approached saturation, the fraction of bound C e l 6 A - C B M 2 a decreased to approximately 10%. Table 3.9 Competition binding summary II: C B M 2 a , C e l 6 A - C B M 2 a and C B M 3 at 4°C in 50 m M potassium phosphate, p H 7.0. CBM* Competitor Cellulose CBM2a K a apparent (106 M"1) Figure C B M 2 a - O G ' (none) P A S C 0.50 (± 0.02) 3.15 C B M 2 a - O G 1 C B M 3 (15 uM) P A S C 0.17 (±0 .02 ) 3.15 CBM2a -6(? (none) P A S C 1.2 (±0 .1 ) 3.16 C B M 2 a - O G 2 Cel6A-CBM2a-AX(15 .8 uM) P A S C 0.39 (± 0.03) 3.16 CBM2a -dC? (none) B M C C 4.3 (±0 .5 ) 3.17 C B M 2 a - O G 3 Cel6A-CBM2a-AX(12 .5 uM) B M C C 1.2 (±0 .1 ) 3.17 *superscript number delineates the CBM2a -OG preparation used in the competition experiment 75 Free (uM) Figure 3.15 Binding of C B M 2 a - 0 G to P A S C in the presence of C B M 3 . Binding of C B M 2 a alone ( • ) and in the presence of 15 p M C B M 3 (O). 76 1.00 0.00 I i i i i • ' I 0 1 2 3 4 5 6 7 8 9 10 Free C B M 2 a - O G (u.M) 1.00 B) 0 . 0 0 H — i — i — i — i — i — i — > — ' — i — i — i — ' — i — > — i — i — 1 — 1 — 1 — I 0.00 0.25 0.50 0.75 1.00 [B]/[N0]CBM2a-OG Figure 3.16 Binding of C B M 2 a - O G to P A S C in the presence of C e l 6 A - C B M 2 a - A X . A ) Binding of C B M 2 a - O G alone ( • ) and in the presence of C e l 6 A - C B M 2 a (O) ; Ce l6A-C B M 2 a - A X bound vs. C B M 2 a - O G free is also plotted ( A ) . B) Fraction bound C B M 2 a - A X ([B]/[N„]) vs. fraction bound C B M 2 a - O G ([B]/[N„]). 7 7 0.00 I i • • • • i i i. i I 0 1 2 3 4 5 6 7 8 9 10 Free CBM2a-OG (uM) o.oo H—i—i—•—•—i—•—.—•—.—i—.—•—i—i—i—•—.—i—•—I 0.00 0.25 0.50 0.75 1.00 [B]/[N0] CBM2a-OG Figure 3.17 Binding of C B M 2 a - O G to B M C C in the presence of C e l 6 A - C B M 2 a . A ) Binding of C B M 2 a - O G alone ( • ) and in the presence of C e l 6 A - C B M 2 a (O) ; C e l 6 A - C B M 2 a - A X bound vs. C B M 2 a - O G free is also plotted ( A ) . B ) Fraction bound C B M 2 a - A X ([B]/[N 0]) vs. fraction bound C B M 2 a - O G ([B]/[N,,]). 78 3.4.8 CBM4-1 and CBM17 competition As mentioned previously, C B M 4 - 1 and C B M 17 have similar substrate specificity binding to cello-oligosaccharides, greater than 5 or 6 glucose units in length and to amorphous cellulose (8, 61, 125, 127); C B M 1 7 , however, binds to P A S C with approximately 4-fold higher affinity than C B M 4 - 1 (Table 3.4). In competition experiments using a constant amount of C B M 4 - 1 - A X as a competitor, the apparent affinity of C B M 1 7 - O G for P A S C was reduced (Table 3.10; Figure 3.18-A). A significant number of the sites bound by C B M 1 7 - O G are also bound by C B M 4 - 1 - A X . However, not all of the C B M 4 - 1 - A X was displaced as the concentration of C B M 17 increased (Figure 3.18-B). In the complementary experiment, the apparent affinity of C B M 4 - 1 - A X was reduced in the presence of a constant amount of C B M 1 7 - O G (Table 3.10, Figure 3.19). Only a small fraction of the C B M 1 7 - O G molecules bound were displaced as the C B M 4 - 1 approached saturation; a majority of the sites that are bound by C B M 1 7 - O G are not recognized by C B M 4 - 1 - A X . The amorphous regions of P A S C are therefore heterogeneous, composed of sites recognized by only C B M 4 - 1 , C B M 1 7 or both C B M s . Table 3.10 Competition binding summary III: C B M 17 and C B M 4 - 1 at 4°C in 50 m M C B M Compet i tor C B M K a apparent (106 M"1) [No] ( u m o l g - cel lulose) F igure C B M 1 7 - O G ( none ) 0.81 (±0 .05) 27.8 (± 0.6) 3.18 C B M 1 7 - O G C B M 4 - 1 - A X 0.31 (±0 .01) 27.8 (± 0.6) 3.18 C B M 4 - 1 - A X ( n o n e ) 0.24 (± 0.05) 7.1 (±0 .4 ) 3.19 C B M 4 - 1 - A X C B M 1 7 - O G 0.003 (± 0.007) 7.1 (±0 .4 ) 3.19 79 B) •3 0.5(H o . o o H — i — i — . — . — i — . — ' — i — i — i — i — i — i — i — i — i — i — i — i — I 0.00 0.25 0.50 0.75 1.00 [B]/[N 0 ] C B M 1 7 - 0 G Figure 3.18 Binding of C B M 1 7 - O G to P A S C in the presence of C B M 4 - 1 - A X . A ) Binding of C B M 1 7 - O G alone ( • ) and in the presence of 15 p M C B M 4 - 1 - A X (O); C B M 4 - 1 - A X bound vs. C B M 1 7 - O G free is also plotted ( A ) . B) Fraction bound C B M 4 - 1 - A X ([B]/[N ( 1]) vs. fraction bound C B M 1 7 - O G ([B]/[N„]). 80 1.00-B) f - 0.50-s m u 0.25-^ 0.00-I . . . . 1 . . . . 1 . . . . 1 . . . . 1 0.00 0.25 0.50 0.75 1.00 CBM4-1 [B]/[N0] Figure 3.19 Binding of C B M 4 - 1 - A X to P A S C in the presence of C B M 1 7 - O G . A ) Binding of C B M 4 - 1 - A X alone ( • ) and in the presence of 15 | xM C B M 1 7 A X (O) ; C B M 1 7 - O G bound vs. C B M 4 - 1 - A X free is also plotted ( A ) . B) Fraction bound C B M 1 7 - O G ([B]/[N„]) vs. fraction bound C B M 4 - 1 - A X ([B]/[N„]). 81 3.5 CBM2a binding adsorption and exchange 3.5 .1 R a t e of A d s o r p t i o n of C B M 2 a to P A S C a n d B M C C C B M 2 a bound to P A S C at a similar rate at the two concentrations tested. At 19.6 LLM, C B M 2 a bound to P A S C with a t 1 / 2 of 4 min; at 27.8 u M , C B M 2 a binds with a t1/2 of 6 min. The rate of adsorption at both concentrations is within experimental and regression standard error (Figure 3.20-A). The rate of C B M 2 a adsorption to B M C C was too fast to measure by this method; C B M 2 a reached binding equilibrium in less than 30 s (Figure 3.20-B). Assuming that C B M 2 a binds to crystalline regions of B M C C and P A S C by a similar mechanism, then the binding of C B M 2 a to P A S C is likely limited by mass transfer through the porous cellulose matrix of P A S C . 3.5.2 Equ i l ib r ium e x c h a n g e of C B M 2 a o n B M C C C B M 2 a binds irreversibly to cellulose (25, 57, 88). Binding exhibits hysteresis; the desorption isotherm, generated from dilution, is not congruent with the adsoprtion isotherm. Two cellulases with family 2 C B M s (11) (Thermomonosporafusca Cel6B, formerly E3 and Cel5A, formerly E5) also show binding hysteresis yet they exhibit surface exchange. The cellulases bound to the surface are displaced by those free in solution, without apparent disturbance of the apparent binding equilibrium. Based on observations from the competition experiments, C B M 2 a also exhibited surface exchange; the extent and rate of the surface exchange of C B M 2 a was investigated. B M C C was loaded with C B M 2 a - A X at a total concentration of 18.2 | i M . After equilibration at 4°C, the average concentration of free C B M 2 a - A X was 4.2 pJVl and the average bound concentration of C B M 2 a - A X was 14.1 uxnol-g"1 (approximately the maximum loading of 82 B M C C with C B M 2 a ) . The concentration of bound C B M 2 a removed during the three washing steps was less than 1 p M , indicating that almost all of the bound C B M 2 a is bound irreversibly. After washing, C B M 2 a - O G was added at different concentrations: below equilibrium (1.4 p M ) , at approximately equilibrium (3.2 p M ) and greater than equilibrium (14 p M ) . After equilibration, the concentration of C B M 2 a - A X and C B M 2 a - O G in solution was measured and the concentration of the labelled species bound to B M C C calculated. The total concentration of C B M 2 a bound to B M C C ( C B M 2 a - A X plus C B M 2 a - O G ) remained constant at approximately 14 pmol-g 1 regardless of the concentration of C B M 2 a - O G added (Figure 3.21). The proportion of the C B M 2 a - A X and C B M 2 a - O G bound to the surface did however depend on the overall proportion of each labelled C B M 2 a present (Table 3.11). For example, at high concentration addition of C B M 2 a - O G (14 p M ) , C B M 2 a - O G represented 50% of the total C B M 2 a present, and 42% of the total C B M 2 a bound to B M C C C B M 2 a - O G . A large fraction of the surface-bound C B M 2 a molecules can therefore exchange with C B M 2 a molecules in the solution phase. The quantification of the extent of exchange is much more reliable at highest concentration of added C B M 2 a - O G ; at lower concentrations (<3.2 p M ) , the concentrations of C B M 2 a - A X and C B M 2 a - O G are difficult to measure with confidence. Table 3.11 Exchange at equilibrium of C B M 2 a bound to B M C C at 4°C in 50 m M potassium phosphate, p H 7.0. CBM2a-OG Added (MM) Sum CBM2a-OG/Total CBM2a Bound CBM2a-OG/Total Bound CBM2a Sum CBM2a-AX/Total CBM2a Bound CBM2a-AX/Total Bound CBM2a 0 (Buffer) 0 0 1 1 1.4 0.09 0.06 0.91 0.94 3.2 0.18 0.15 0.82 0.85 14.0 0.50 0.42 0.50 0.58 Note: The Sum C B M 2 a - O G equals the total C B M 2 a - O G present (sum of free C B M 2 a - O G and bound C B M 2 a - O G ) , and similarly C B M 2 a - A X equals the total C B M 2 a - A X present. Total bound C B M 2 a equals the sum of bound C B M 2 a - A X and bound C B M 2 a - O G . 83 3.5.3 R a t e of C B M 2 a e x c h a n g e o n B M C C The rate that surface exchange occurs was investigated. Ten samples, each with 1 mg of B M C C , were loaded with a total of 16 | i M of C B M 2 a - A X and equilibrated at 4°C. At equilibrium, the average bound C B M 2 a - A X was 12.5 pmol-g' , the average free C B M 2 a - A X was 3.4 p M . Each sample was washed three times with potassium phosphate buffer. C B M 2 a -O G was then added and incubated with B M C C for different times, the amount of C B M 2 a -A X and C B M 2 a - O G remaining in the solution phase was measured and the concentration of each labelled species bound to B M C C was calculated. C B M 2 a - O G re-established equilibrium by exchanging with the C B M 2 a - A X bound to the cellulose surface, in less than 30 seconds, faster than could measured by this method (Figure 3.22). 84 Time (min) Figure 3.20 Adsorption kinetics of C B M 2 a binding to P A S C and B M C C . A ) Rate of adsorption to P A S C (fraction of equilibrium bound; [B] / [B m J vs. time (min)) at a total concentration of 19.6 u M C B M 2 a - O G ( • , solid line) and 27.8 u M C B M 2 a - O G ( O , dashed line). B ) Rate of C B M 2 a - O G adsorption to B M C C at a total concentration of 19.7 u M ( • ) 85 & 4 H 0 1 ^ E s U " I - o - B o u n d CBM2a-OG Tota l B o u n d CBM2a o u n d -  B o u n d CBM2a-AX Free CBM2a (|iM) Figure 3.21 Surface exchange of C B M 2 a , at equilibrium, on B M C C . Concentration of C B M 2 a populations versus the total free concentration of C B M 2 a ; C B M 2 a - O G ( O , dashed line), C B M 2 a - A X ( • , dotted line) and total C B M 2 a (a sum of bound C B M 2 a - A X and C B M 2 a - O G ; • , solid line) is plotted; the connecting lines are added for visual reference only. F igure 3.22 Kinetics of C B M 2 a surface exchange on B M C C . Bound C B M 2 a populations C B M 2 a - A X ( O , dashed line), C B M 2 a - O G ( • , dotted line) and total C B M 2 a (a sum of bound C B M 2 a - A X and C B M 2 a - O G ; #, solid line) vs. time are plotted; connecting lines are added for visual reference only. 86 3.6 CBM2a-cellulose interaction observed by solid state NMR This study is the first documented attempt to directly observe the CBM-cel lulose interaction using solid state N M R . Detection of this interaction relies upon observing interacting nuclei that are visible by N M R , such as 1 3 C and l 5 N . Here, 1 3C-enriched cellulose was produced using A. xylinum and C B M 2 a was homogeneously labelled with l 5 N ( l 5 N - C B M 2 a ) . 3.6.1 P r o d u c t i o n a n d charac te r i za t ion of 1 3 C - e n r i c h e d bacter ia l c e l l u l o s e A sample of C B M 2 a bound to bacterial cellulose was prepared for the solid state N M R studies using cellulose produced by A. xylinum and processed as described in a previous section (Section 2.6.2). The C B M 2 a had binding affinity and capacity ( K a = 1.7 x 10 6 M ' 1 , [N 0] = 12.9 pmol-g"1) for binding this preparation of bacterial cellulose that was similar to C B M 2 a binding to B M C C . 1 3C-4-enriched bacterial cellulose, synthesized from D-glucose-4 1 3 C in minimal medium, yielded 25 mg of cellulose (dry weight) from 100 ml of culture. The C-4 of the bacterial cellulose was not labelled completely uniformly or uniquely. Signals corresponding to C - l and C-2,3,5 regions of cellulose were evident in the spectrum of 1 3 C-4-enriched cellulose (Figure 3.23). Through unanticipated metabolic processes, the bacteria scrambled some of the label that was incorporated into the bacterial cellulose. Since the bacterium rearranges the 1 3 C label from glucose, A. xylinum was grown in a rich medium using glycerol, because, the 1 3C-labelled glycerol preparations are significantly less expensive than 1 3C-glucose preparations. Growing A. xylinum 53524 in H & S medium, using glycerol-1,3- 1 3C 2 , resulted in higher cellulose yields than in minimal medium, (191 mg / 100 ml of culture, dry weight) with significant labelling (-50%) at C - l , C-3, C-4 and C-6 (Figure 3.24). The main advantage of the labelled cellulose preparations is the greatly improved signal to noise ratio as evidenced by the much fewer number of scans needed to collect a 87 high resolution spectrum. Using the 1 3 C natural abundance (-1.1%), the spectrum of bacterial cellulose was the sum of 40 000 scans; spectra of 13C-enriched cellulose, with comparable resolution, could be collected with only 400 scans (Figure 3.24). It is very unlikely that CBM-cel lulose interaction could be detected by relying on 1 3 C natural abundance alone. 3.6.2 C B M 2 a - c e l l u l o s e interact ions Approximately 7 mg of 1 5 N - C B M 2 a was bound, in solution, to -50 mg (dry weight) 1 3 C -enriched cellulose from the glycerol-1,3- 1 3 C 2 synthesis; excess solution was removed so that the sample was approximately 40-60% water content, by weight. The interaction between the l 5 N nuclei of C B M 2 a and l 3 C of the cellulose should be detectable by a CP-drain experiment if there are a sufficiently large number of 1 3 C / 1 5 N interactions for the sensitivity of the experiment to detect. The CP-drain experiments rely upon observing the C P / M A S spectrum of one nucleus (e.g. I 3 C of the enriched cellulose), after transfer of magnetism to a second nucleus (e.g. 1 5 N of 1 5 N - C B M 2 a ) . If the two nuclei are in close proximity (i.e. the l 5 N of the C B M bound to the surface of the 1 3C-labelled cellulose), there wi l l be transfer and a decrease in signal wi l l be observed; if they are too distant, no transfer wi l l occur. This experiment is relatively robust and since it measures a decrease in the 1 3 C signals caused by only the interactions with the 1 5 N nuclei, it should therefore unambiguously detect only surface binding of 1 5 N - C B M 2 a to l 3C-enriched cellulose. Moreover, nuclear interactions between specific 1 3 C atoms of the cellulose (eg. C-4), and specific 1 5 N can be distinguished, in theory. Unfortunately, we did not detect CBM-cellulose interaction by the CP-drain experiments; the spectrum of the labelled cellulose after CP-drain was virtually indistinguishable from the pre-CP-drain spectrum (shown in Figure 3.24-B). The system was working properly as the l 5 N -88 C P / M A S spectrum of I 5 N - C B M 2 a bound to bacterial cellulose could be measured and collected (Figure 3.25). This is the first time a C B M has been observed bound to cellulose. The 1 5 N - C P / M A S spectrum can be approximately correlated with a one-dimensional projection of the l 5 N signal from the H S Q C of 1 5 N - C B M 2 a (Figure 3.25); the spectra are somewhat similar yet, characteristically, the peaks of the 1 5 N - C P / M A S spectrum are broader than that of the spectrum measure of C B M 2 a in solution. C B M 2 a itself appears to be folded when bound to the surface and is not denatured. 89 ( A ) B a c t e r i a l C e l l u l o s e , C-2,3,5 , C-4 115 110 105 100 95 90 85 80 75 70 65 60 55 ppm ( B ) 1 3 C - e n r i c h e d B a c t e r i a l C e l l u l o s e 115 110 105 100 95 90 85 80 75 70 65 60 55 Ppm Figure 3.23 1 3 C - C P / M A S Spectra of bacterial cellulose and 1 3C-enriched cellulose from 1 3 C-4-glucose synthesis. (A) Bacterial cellulose ( l 3 C natural abundance, 1.1%) spectrum was the sum of 40 000 scans, (B) 1 3C-enriched bacterial cellulose spectrum is the sum of 400 scans. Horizontal bars indicate the spectral regions of the corresponding carbon atoms in the glucose monomer unit of cellulose. 90 ( A ) B a c t e r i a l C e l l u l o s e , C-2,3,5 { I —— 1 S / 1 I u 115 110 105 100 95 90 85 80 75 70 65 60 55 PP™ ( B ) 1 3 C - e n r i c h e d B a c t e r i a l C e l l u l o s e 120 115 110 105 100 95 90 85 80 75 70 65 60 55 Ppm Figure 3.24 C P / M A S Spectra of bacterial cellulose and 1 3C-enriched cellulose from l ,3- ' 3 C-glycerol synthesis. (A) Bacterial cellulose ( 1 3 C natural abundance, 1.1%) spectrum was the sum of 40 000 scans, collected as described in the materials and methods (B) l 3C-enriched (-50%) bacterial cellulose spectrum is the sum of 400 scans. 91 A B 132.0 123.0 114.0 N 15 ( p p m ) vs. liquid ammonia c X 5 O XlO X30 X20 XXQ XOO 90 SO ~70 SO SO IO 30 1=1?'" v s . l i q u i d a m m o n i a Figure 3.25 1 5 N C P / M A S spectrum of 1 5 N - C B M 2 a bound to insoluble cellulose (A) and H S Q C spectrum of of 1 5 N - C B M 2 a in solution (B). The scales of the solid (A) and liquid spectra have been aligned. Peaks of ' 5 N - C P / M A S of 1 5 N - C B M 2 a bound are roughly aligned to the corresponding peaks of the H S Q C peaks of l 5 N - C B M 2 a in solution. A l l spectra are referenced using liquid ammonia; the 1 5 N - C P / M A S spectra had a secondary reference of solid ammonium chloride. The full 1 5 N C P / M A S of , 5 N - C B M 2 a , showing upfield spectral features is shown in (C). 92 4. Discussion The purpose of this study was to further elucidate the nature of the interaction of C B M s with their ligand and in doing so, further understand the biological role that C B M s play as a components in polysaccharide degradation systems and investigate the diversity of C B M structure and function. C B M specificities for the binding to B M C C and P A S C was studied by a series of competition experiments using six C B M s representing the three C B M types. As a consequence of knowing the binding specificities of this complement of C B M s , insights as to the structure of the complex structure of P A S C were gained using the competition binding technique. It was shown that P A S C , has distinct regions of both crystalline and amorphous cellulose. Family 2a C B M s are the most common C B M found in the cellulases and xylanases of C. fimi. The binding of C B M 2 a to cellulose was investigated in more detail to further understand its function in substrate binding, as component of an organism's polysaccharidases, and for the potential enhancement of properties useful in specific applications such as affinity purification and enzyme immobilization. Although it has been reported that C B M 2 a binds with apparent irreversibility, during the course of the competition experiments, it was observed that C B M 2 a molecules that are bound to cellulose exchange with free C B M 2 a molecules in solution. The extent and kinetics of surface exchange was investigated. Knowing that C B M 2 a binds to crystalline cellulose, the residues that are important determinants of binding affinity to B M C C were explored by site-directed mutation. A number of residues on the binding face of the molecule were explored: conserved tryptophan residues were mutated to tyrosine, phenylalanine and alanine; other polar residues with the potential to interact with cellulose were substituted with alanine. 93 For the first time, the direct observation of CJ3M2a bound to insoluble cellulose was attempted using the technique of solid state N M R . l 5 N-labelled C B M 2 a was produced, bound to 1 3C-enriched cellulose and observation was attempted by a CP-drain experiment, a robust and sensitive technique that can detect nuclei that are in close proximity. Further refinement of solid state N M R technique should yield more structural information about C B M 2 a bound to cellulose in its native state. 4.1 Cellulose and CBMs: a diversity of structure and function 4.1.1 B i n d i n g speci f ic i ty of C B M s for c e l l u l o s e The competition binding isotherm experiments, detailed in this study, are a new approach for the investigation of C B M binding specificity for complex substrates. Solution-dependent methods, such as binding studies with soluble ligands (123), N M R , or crystallization of C B M with ligand, are not applicable to studying the specficities of C B M s for complex insoluble substrates such as P A S C . As a consequence of knowing the binding specificity of C B M s , competition experiment can be used to gain information about the structures, and mixtures of structures, present in complex substrates. For example, as the current work shows, P A S C is comprised of different forms of amorphous cellulose as well as a large surface area of crystalline cellulose. This technique could be applied to characterize surfaces the of other substrates, such as wood pulp. 4.1.1.1 CBM2a binds to crystalline regions of cellulose Unti l now, there has been little direct evidence that C B M 2 a binds strictly to the crystalline regions of insoluble cellulose. In competition with type B C B M s with specificities for amorphous cellulose and soluble ligands, it was shown that C B M 2 a binds to sites not 94 recognized by the type B C B M s (79). The type B C B M s used in competition experiments, C B M 4 - 1 and C B M 17, bind soluble oligosaccharides and amorphous cellulose; they have no significant affinity for highly crystalline B M C C (8, 56, 60, 61, 108, 125). C B M 4 - 1 binds saccharide chains within an obvious binding groove, and has the greatest affinity for cellopentaose, or longer oligosaccharides, and amorphous cellulose. Binding is enthalpically driven and is fully reversible (125). C B M 1 7 also binds reversibly to cello-oligosaccharides and amorphous cellulose. The shallow, twisted binding groove of C B M 17 binds cello-oligosaccharides and has the greatest affinity for cellohexaose and P A S C . In contrast to these two modules, the type A module C B M 2 a has a more planar binding face and does not bind small sugars or cello-oligosaccharides. C B M 2 a has a similar affinity for all forms of insoluble cellulose tested, B M C C , Avice l and P A S C ( K a ~10 6 M " 1 ; Table 3.1) suggesting that it binds to the same component in each of these celluloses. Binding is entropically driven, presumably due to dehydration of apolar surface residues (25). Since the binding of C B M 17 and C B M 4 - 1 to P A S C was not significantly affected by the presence of C B M 2 a , and the binding of C B M 2 a to P A S C is largely unaffected by the presence of C B M 4 - 1 , C B M 2 a must bind to regions not recognized by either C B M 4 - 1 or C B M 17. Presumably, C B M 4 - 1 and C B M 17 bind to individual cellulose molecules in amorphous regions of P A S C , whereas C B M 2 a binds to crystalline surfaces (Figure 4.1). Like C B M 2 a , the type A binding modules C B M 3 and C e l 6 A - C B M 2 a , also bind to highly crystalline celluloses , such as B M C C (Table 3.4), but not to cello-oligosaccharides or soluble sugars. A l l three C B M s have a linear platform of aromatic residues exposed on one face of the module that are major mediators of the binding interaction. C B M 2 a and Cel6A-C B M 2 a have very similar amino acid sequences, including three exposed tryptophans on the binding face. They have similar affinities for insoluble cellulose (Table 3.4). Type A C B M s 95 all had a similar capacity for P A S C (-15 pmol-g 1) and a similar capacity for B M C C (-11 pmol-g') (Table 3.4). As might be expected, C B M 2 a binds to the same sites on B M C C and P A S C as C e l 6 A - C B M 2 a . (Figures 3.16 and 3.17; Table 3.9). Although the C B M 3 amino acid sequence is dissimilar to those of the family 2a binding modules, it has a similar anti-parallel [3-sheet structure with a platform of conserved exposed aromatic amino acids. Competition experiments show that C B M 3 binds to the same sites of P A S C as C B M 2 a (Figure 3.15; Table 3.9). The binding of C B M s with affinity for crystalline cellulose is a function of the total solvent exposed surface area of the crystalline cellulose that is available to C B M 2 a . The binding capacity, [N 0 ], does not directly correlate with the bulk crystallinity of the cellulose. Overall, B M C C has a larger proportion of crystalline cellulose than P A S C ; however, based on the larger capacity of P A S C to bind C B M 2 a , a much larger surface area of the crystalline regions of P A S C is available to the binding module and, as shown by competition binding experiments, C B M 2 a binds exclusively to the crystalline regions of cellulose. Paradoxically in C P / M A S spectrum of P A S C (Figure 3.2), the predominant broad C-4 peak at 81-83 ppm, characteristic of disordered, surface exposed C-4 atoms, and the extremely small peak at -89 ppm arising from the highly ordered crystalline regions (4), shows that P A S C , an acid-swollen preparation of Avice l , has significant amorphous characteristic, yet C B M 2 a , which binds to crystalline cellulose, binds to P A S C with high capacity (Table 3.1). On the basis of weight, the crystalline surface area made available to C B M 2 a is increased by approximately six-fold by the acid swelling process that converts Avice l to P A S C . This process is accompanied by with a drastic decrease in the bulk crystallinity, or total volume of the crystalline regions, as characterized by the disappearance of the sharp downfield C-4 peak at -89 ppm in the 1 3 C - C P / M A S spectrum of P A S C compared with Avicel(Figure 3.2). The acid 96 swelling process partially dissolves the cellulose; the cellulose is re-precipitated by dilution in dH 2 0 (37, 139). Cellulose longer than seven glucose monomer units is not very soluble in water and forms aggregates. The crystallinity of P A S C can be envisioned to be a large number of microcrystalline regions formed by the aggregation of a number of cellulose chains. These microcrystalline regions have sufficient surface area to be bound by C B M 2 a , are extremely numerous, manifested in the increased capacity for C B M 2 a , yet are extremely small in volume so are not seen in bulk measurements of crystallinity. Crystalline cellulose may also be heterogeneous, in terms of binding specificity (16, 25). Although I did not detect such heterogeneity, there is some evidence, based on ITC data (25), that crystalline cellulose presents two classes of high affinity binding sites to C B M 2 a . The affinities of C B M 2 a for the crystalline regions of B M C C , P A S C , and Avice l are similar; the relatively small differences (Table 3.1) likely reflect the proportion of high and low affinity binding sites on each cellulose preparation. The structural differences between these two apparently different binding regions are unclear. It could be a combination of the relative proportion of the cellulose allomorphs, such as cellulose I a , Ip and cellulose II. Further competition experiments, like those performed in this study, involving an expanded library of type A C B M s binding to cellulose with different proportions of cellulose allomorphs may reveal further details of the complex surface structure of crystalline cellulose, and the basis for C B M s distinguishing the various sites. 97 Figure 4.1 C B M s bound to native cellulose. A ) Native cellulose with crystalline and amorphous regions. Individual cellulose molecules are represented by black lines. The cellulose molecules are aggregated into crystalline regions, which are highly ordered, and more disordered amorphous regions with semi-soluble, single cellulose chains. The reducing ends of the parallel cellulose molecules are also indicated. B) C B M s bound to cellulose. C B M 2 a (grey-shaded) and C B M 3 (light-grey) bind to the crystalline regions, C B M 9 - 2 (unshaded) binds to the available reducing ends and C B M 4 - 1 (chequered) and C B M 17 (cross-hatched) bind to amorphous regions. A s demonstrated by competition binding experiments, some of the sites of amorphous cellulose are bound only by C B M 17 or C B M 4 -1; some amorphous cellulose sites can be bound by both. 4.1.1.2 Heterogeneity of amorphous cellulose structure The two type B binding modules, C B M 4 - 1 and C B M 17, bind cello-oligosaccharides and amorphous cellulose; they compete for binding sites on P A S C . The apparent affinity of CBM4-1 for P A S C is reduced from 0.24 x 106 M " 1 to 0.003 x 106 M 1 in the presence of C B M 17 (Figure 3.19, Table 3.10). Similarly, the apparent affinity of C B M 17 for P A S C is reduced from 0.81 x 10 6 M _ I to 0.31 x 10 6 M~* in the presence of C B M 4 - 1 (Figure 3.18, Table 3.10). There are, however, selected regions of the cellulose that are recognized uniquely by one and not the other C B M . The fraction of C B M 4 - 1 bound ([B]/[N 0]) decreased to only -0.5 as C B M 1 7 approached saturation (Figure 3.18-B) and only a small fraction of the bound 98 C B M 1 7 was displaced by saturating amounts of C B M 4 - 1 (Figure 3.19-B). The data suggest that there are at least three distinct sites on P A S C , one recognized only by C B M 4 - 1 , one recognized only by C B M 1 7 , and a third class of sites recognized by both of them. The structures of the binding sites on C B M 17 and C B M 4 - 1 may help to reveal the differences between these sites on P A S C (Figure 4.2). In the crystal structure of C B M 4 - 1 bound to cellopentaose, binding occurs in a relatively narrow groove on the polypeptide; the sugar chain is almost fully encompassed by the module. The C B M contacts both faces of the cello-oligosaccharide molecule. B y contrast, the binding groove of C B M 17 is much shallower, somewhat twisted and makes contact with only one face of the sugar chain. In a preparation of P A S C with a number of complex amorphous structures, C B M 4 - 1 therefore could bind only fully accessible individual cellulose molecules, whereas, C B M 17 could bind to some of the fully accessible chains recognized by C B M 4 - 1 , as well as some partially accessible single molecules. The capacity of P A S C is approximately three times greater for C B M 17 than for C B M 4 - 1 , suggesting a much larger proportion of amorphous cellulose comprises only partially accessible molecules. These subtle differences in the specificity of C B M 17 and C B M 4 - 1 underline the heterogeneity of amorphous cellulose in P A S C (Figure 4.1; Figure 4.2). 99 Figure 4.2 Details of the binding sites of C B M 4 - 1 (A) and C B M 17 (B) with bound cellopentaose and cellotetraose, respectively. The cellooligsaccharides are rendered in ball and stick form. To emphasize the shape of the respective ligand-binding sites, the protein surface is rendered to display the solvent-accessible surface; it is shaded according to the electrostatic charge. In both figures, the structure of the complex is viewed parallel to the long axis of the cellooligasaccharide (A. B . Borason, V . Notenboom, A . Freelove, unpublished results) 100 4.1.2 C B M s : a g e n t s for target ing p o l y s a c c h a r i d a s e s a n d for sa tura t ing s u b s t r a t e Cellulose can have different crystalline structures (2, 3). For example, cellulose I, the form of cellulose produced by cellulose synthase, comprises a mixture two distinct crystalline types, designated I a and I p , the proportion of each type depending on the source. These competition experiments show that amorphous cellulose is also heterogeneous. In the plant cell wall, cellulose is intimately associated with hemicellulose which may include mannans, galactomanans, xylan, xyloglucans, pectin and some glucans (Figure 1.1). Mirroring the diversity of cellulose and polysaccharide polymers, various organisms have evolved an array of modular polysaccharidases to degrade them. The C B M s display an equally diverse array of substrate specificities. Currently, among the C B M s that have been characterized, are those that bind cellulose (9), xylan (20, 112), and mannan (119). Based upon overall architecture and substrate specificity, there are three types of C B M . Within each type, and often within C B M families, the binding specificities of individual C B M s are different. C B M s are thought to promote the hydrolysis of polysaccharides by increasing the concentration of enzyme on insoluble and semi-soluble substrates (38, 47, 98, 128) or, in the case of T f C e l 9 A - C B M 3 (103), by guiding substrate into the active site of the catalytic module. C B M s could also promote substrate hydrolysis by targeting their associated hydrolases to specific sites on the substrates, especially in heterogeneous material like the plant cell wall . The specificity of the C B M can influence the overall specificity and activity of the associated cellulase. For example, when the family 2a C B M from CfCe l6A (CenA) was exchanged for the tandem family 4 C B M s of CfCel9B (CenC), the resulting enzyme, with two C B M 4 - 1 modules and the Cel6A catalytic module, was less active on Avicel and B M C C than Ce l6A, yet more active on C M C , a soluble derivative of cellulose, and cellulose azure, a regenerated form of cellulose (22). In this case, the specificity or enzyme activity of 101 the catalytic module itself is not changed, however the types of cellulose for which the enzyme is most active changes and this change is mediated by the associated C B M . The specificity of the C B M may, or may not, correlate with the overall substrate specificity of the associated enzyme. The binding specificities of C B M 2 a modules do not correlate with the associated enzyme's substrate. In C.fimi, C B M 2 a modules are associated with enzymes that cleave p-1,4-linked glycans: cellobiohydrolases (Cel48A and Cel6B) (80, 107), endoglucanases with high activity with crystalline (Cel9A; Cel5A) or amorphous cellulose and soluble cellulose derivatives (Cel6A, Cel9A, Cel9B), or xylan ( X y n l O A ) (127). A l l of the Family 2a C B M s studied bind to crystalline cellulose only. There is some evidence that binding specificity of family 4 C B M s does correlate with overall enzyme substrate specificity (144). Among the polysaccharidases with family 4 C B M s are three cellobiohydrolases (35, 133, 143), a xylanase (62), P-l,4-glucanases (55, 127), and a laminarinase (P-l,3-glucanase) (145). The family 4 C B M s from C.fimi Cel9B, as discussed previously, bind to amorphous cellulose and soluble sugar polymers. Cel9B itself has activity on amorphous cellulose. Unlike C f C e l 9 B - C B M 4 - l and C f C e l 9 B - C B M 4 - 2 , the family 4 binding modules from Thermotoga neopolitana laminarinase (Laml6A) , potentially members of a sub-family within family 4, do not bind P-l,4-glycans; they have affinity for soluble (TnLam 16A-CBM4-1 and TnLam 16A-CBM4-2) and insoluble (TnLam 1 6 A - C B M 4 -2) 1,3-P-glucans. The binding specificities of these two binding modules is virtually identical to the preferred substrates of the catalytic module (144). More information about the binding specificities of other family 4 C B M s and their associated glycosyl hydrolases is neccessary to unequivocally establish a correlation. 102 There are eight polysaccharidases with C B M 17 modules and each is associated with a family 5 glycosyl hydrolase. Only three of the enzymes, and one associated C B M 17, have been characterized. The endo-l,4-glucanase from Bacillus sp. 22-28 is active with C M C (83); the endo-l,4-glucanase from Bacillus sp. KSM-S237 also has activity with C M C , it does not hydrolyze crystalline cellulose (46). The binding characteristics of the associated C B M 17s are not known. The C. cellulovorans cellulase 5A (EngF) has the greatest activity with C M C , followed by P A S C and lichenan. Little or no activity was observed with laminarin, xylan and Avice l were tested as substrates (108); the binding module also binds to C M C and amorphous cellulose. With only these examples, there is not enough information to assess any apparent correlation between C B M 17 binding specificity and its associated enzyme activity. In many cases, there is little apparent correlation between C B M binding specificity and enzyme substrate specificity, it is unlikely that C B M s act only to target the enzyme to a particular substrate. C B M s could be increasing the efficiency of the degradation of insoluble, and heterogeneous substrates (Figure 1.1) by binding to one area of the complex substrate while the catalytic module hydrolyzes substrate in an area located away from the site bound by the C B M . For example, the family 2a binding module of CfCe l6A binds to a crystalline region of cellulose while the enzyme, separated from the C B M by a linker sequence, may access available amorphous regions of the cellulose (see Figure 4.1; crystalline and amorphous cellulose regions are adjacent). Polysaccharides are intimately associated in the plant cell wall, the C B M 2 a of C f X y n l O A may bind crystalline cellulose, while the associated catalytic module digests xylan that is within close proximity. C B M s , therefore, may also ensure adequate coverage of the complex substrate by enzymes with different specificities. 103 4.2 Adsorption of CBM2a to cellulose Six of the eight cellulolytic enzymes of C.fimi have family 2a C B M s (Figure 1.2). This module is an important component of the polysaccharidases of system of this organism. Additionally, as a fusion partner, C B M 2 a has been used as an affinity tag for the purification or immobilization of a number of polypeptides (124). Both an understanding of C B M 2 a in its biological context and for the alteration or customization of C B M 2 a for specific applications motivate the need to have more detailed understanding of the specific interactions of C B M 2 a with cellulose. To do so, the binding interaction of C B M 2 a with B M C C and P A S C was characterized, the irreversibility of binding to B M C C was assessed, the amino acids important in binding were probed and direct observation by solid state N M R C B M 2 a bound to cellulose was attempted. 4.2.1 Irreversibility of a d s o r p t i o n of C B M 2 a It has been reported that C B M 2 a adsorbs irreversibly to insoluble cellulose (25, 87); complete removal of C B M 2 a from the solution phase does not result in any appreciable desorption of C B M 2 a from the surface. Binding exhibits hysteresis; the isotherm that describes the adsorption of C B M 2 a to cellulose is not equivalent to the desorption isotherm generated by of removal of C B M 2 a from solution. The equilibrium described by the adsorption isotherm is not re-established. Although apparently irreversible, binding is dynamic: under conditions where all C B M 2 a in the solution phase has been removed, C B M 2 a moves in two-dimensions on the surface without desorbing from it (57). Paradoxically, the current work shows that a high percentage of C B M 2 a molecules bound to the cellulose surface can exchange with C B M 2 a molecules in solution; binding is not strictly irreversible. In these exchange experiments, most (>93%) of C B M 2 a - A X was not desorbed from B M C C by incubation with buffer alone however, any addition of free C B M 2 a - O G 104 displaced C B M 2 a - A X bound to the surface. Most of the C B M 2 a molecules bound to B M C C are available to exchange. The process of surface exchange of C B M 2 a is rapid. Binding equilibrium is re-established in less than 30 s (Figure 3.22) faster than the rate of exchange observed for the family 1 C B M , T r C e l 7 A - C B M l (73). The adsorption of C B M 2 a , and other binding modules, can be described by the Langmuir-like binding expression ((37) and equation 1), but the observed binding hysteresis when protein is removed from the solution phase and the surface exchange indicates that the binding reaction is not a simple two-state equilibrium. The actual mechanism of surface exchange is not known, however, the data is consistent with a model requiring the interaction of three components: C B M 2 a in solution, C B M 2 a bound to the surface, and the sorbant surface itself. C B M 2 a in the solution phase is required to displace of C B M 2 a bound to the surface, indicating a protein-protein interaction drives the direct, one to one exchange of bound and free C B M 2 a . This process can be envisioned using the following speculative model (Figure 4.3): 1 o Q Q '7777////////777// 2 S/777////////777// 3 o '/777////////777// 4 //77////77///777// 5a 5b O - O O Q O O '/777/////////7T// S/777////////777// Figure 4.3 A model of C B M 2 a surface exchange. C B M 2 a establishes an apparent adsorption equilibrium (1). C B M 2 a in the solution phase is removed, bound C B M 2 a does not desorb because of the energy barrier involved with the hydration of the tryptophan-rich binding face. C B M 2 a added to the solution phase (3). B y diffusion, solution-phase C B M comes in contact with bound C B M and the surface and there is a protein-protein interaction (4) with two possible results: free C B M 2 a binds to the surface, bound C B M 2 a released into the solution phase (5a) or free C B M 2 a separates from bound C B M 2 a without displacement of the bound C B M 2 a (5b) 105 A C B M 2 a molecule is bound to the surface of cellulose; it does not desorb from it when C B M 2 a in the solution phase is removed. C B M 2 a added to the solution diffuses to the surface and is involved, in a three-way interaction involving the bound C B M 2 a , the free C B M 2 a and the surface itself. There are two possible outcomes of this interaction. The bound C B M 2 a is displaced, and the formerly free C B M 2 a binds to the surface, resulting in a one-to-one exchange of bound and free C B M 2 a . Or, the free C B M 2 a dissociates from the bound C B M 2 a and the surface without exchanging. The nature of the binding interaction of C B M 2 a and the cellulose surface could aid in explaining the mechanism of the protein-protein-surface interaction resulting in exchange. Dehydration of the tryptophan-rich hydrophobic binding ridge drives binding; the desorption of C B M 2 a directly into solution is prevented by the energy required to rehydrate the hydrophobic binding face. Exchange or displacement requires a protein-protein interaction, perhaps involving contact of two C B M 2 a hydrophobic binding faces. This protein-protein, interaction may be a lower energy, intermediate step between C B M 2 a bound to the surface and C B M 2 a free in solution that allows for the desorption of one C B M 2 a molecule, with the concomitant adsorption of the second C B M 2 a molecule. C B M 2 a is not unique in its apparent irreversibility of binding and ability to exchange. Cellulases with either family 1 (69) or family 2 C B M s (11) also demonstrate surface exchange. Of two Thermomonosporafusca cellulases with family 2a C B M s (Cel6B, formerly E3 and Cel5A, formerly E5), only about 75% of the bound population of these cellulases were available to exchange, whereas most of the C. fimi C B M 2 a molecules bound to B M C C were available for surface exchange (Table 3.11). Additionally, there is no obvious correlation between the apparent binding irreversibility, and surface exchange (11, 69, 73); C B M s that bind reversibly or irreversibly demonstrate surface exchange. 106 There are a number of biologically relevant reasons for C B M s and cellulases in solution to have the ability to exchange with those bound to the surface of insoluble substrates. Exchange may ensure that enzymes bound to the surface of substrate, but no longer functional, are continually turned over and replaced by functional enzymes. Exchange might be a mechanism for swapping one for an enzyme with another having different catalytic specificity. For example, plant material is heterogeneous, so it is possible that if one enzyme has locally exhausted its substrate in a particular area, it is replaced with another enzyme, with different specificity. Efficient and complete degradation of an heterogeneous substrate is ensured. Some of the cellulases (Cel9A, Cel5A, Cel6B, and Cel48A) produced in cultures of C.fimi grown on Avice l are subject to proteolysis, resulting in the separation of the C B M from the catalytic module (105). Surface exchange could assist the replacement of a substrate-bound, non-catalytically productive C B M , resulting from proteolytic cleavage, by an intact and active cellulase. 4.2.2 A m i n o a c i d r e s i d u e s invo lved in the b ind ing of C B M 2 a to crysta l l ine c e l l u l o s e The single-site mutation studies indicate that binding of C B M 2 a involves the concerted interaction with crystalline cellulose of a cluster of solvent-exposed residues located on a planar surface region (Figure 3.4) defined by the presence of three tryptophan residues (W17, W54, and W72). Each of the tryptophan residues makes a significant but different contribution to binding. W 7 2 A has 15-fold lower affinity for B M C C compared to the wild-type C B M 2 a . Replacement of W72 with either phenylalanine or tyrosine, however, results in relatively little loss of binding affinity, suggesting that the contact formed between W72 and the sorbent surface may involve hydrophobic interactions between the side chain and the apolar face of a glucopyranoside ring within the crystalline cellulose matrix. 107 The 100-fold loss in affinity exhibited by W 5 4 A is an order of magnitude more than for any other mutant and reflects the essential role of this residue in binding. The more conservative substitution of W54 with either phenylalanine or tyrosine reduces binding affinity only 10-fold. Thus, like W72, W54 appears to couple to the crystalline cellulose matrix, at least in part, through favorable hydrophobic interactions. The importance of the apolar aromatic character of W54 and W72 therefore supports ITC studies (25) which indicate that binding is primarily driven by dehydration of residues in close contact with the crystalline cellulose surface. Dehydration of contacting sorbent and protein surfaces and the concomitant formation of strong van der Waals contacts are known to drive a wide range of specific and non-specific protein adsorption processes (48). As observed for binding of C B M 2 a to B M C C (25), the thermodynamic signature of an adsorption process dominated by dehydration effects is a large positive change in entropy and a large negative change in heat capacity, both of which are due to the release of ordered water in the first and second solvation shells. Replacement of the tryptophan at position 54 with phenylalanine leads to 10-fold drop in affinity, in contrast to W72F which has only a two-fold reduction from the wild-type. This suggests that the aromatic hydrophobic characteristic of W54 is essential for favourable interaction with the crystalline cellulose surface. Both W54, through its pyrrolic amine, and the cellobiose repeating unit of cellulose have the potential to form hydrogen bonds. The arrangement of [3-linked glucopyranoside units in cellulose presents a uniform surface distribution of hydroxyl groups on the outside of each molecule. In native crystalline cellulose, the rigid (3-1,4 glycosidic chain linkage positions these hydroxyl groups to allow all but one of the hydrogen bonds (per glucopyranoside) to be satisfied at both the (110) and (1-10) crystal faces through in-plane inter-chain interactions, (see Figure 4.4). Thus, in the 108 absence of local structural perturbation, cellulose I appears to offer a suitable proton acceptor for hydrogen bond formation with the pyrrolic amine of W54. The potential to form an intermolecular hydrogen bond suggests that W54 may provide binding specificity in addition to contributing to the overall driving force for adsorption to the crystalline cellulose surface due, in large part, to dehydration of the hydrophobic indole ring and the underlying crystalline cellulose surface, which exhibits a pronounced hydrophobic character. The importance to binding affinity of proper formation of an intramolecular hydrogen bond involving the pyrrolic amine of a surface tryptophan is suggested by mutants of W17 and N15. Replacement of the tryptophan at position 17 with either alanine or phenylalanine, neither of which have hydrogen bonding potential, results in an order of magnitude reduction in binding affinity. A n equivalent reduction in affinity is observed for N 1 5 A . The similarity in the affinities of both of these mutants could be due to the necessity of an intramolecular hydrogen bond, likely involving an essential bridging water molecule between N15 and the pyrrolic amine of W17, locking W17 into a specific orientation. This argument is supported by the fact that the mutant W 1 7 Y maintains native-like affinity, presumably because the intramolecular hydrogen bonding requirement for proper orientation of the side chain is at least weakly satisfied by the phenolic group of tyrosine. Collectively, the properites of the site-directed mutants suggest that effective binding of a family 2a C B M to crystalline cellulose requires an aromatic group at position 72, a tryptophan at position 54, and either tyrosine or tryptophan, for possible hydrogen bond formation, at position 17. The tryptophan residues are well conserved among members of family 2a, consistent with the results obtained here by site specific substitution. W72 is the least well conserved of the three surface tryptophans. In 6 of approximately 45 members, this position is occupied by tyrosine. The interchangeability of tryptophan and tyrosine at this 109 position in C B M 2 a , also mirrored in other family 2a members, reinforces the role of hydrophobic interaction of an aromatic ring with the glucopyranoside rings of the cellulose surface. W54 is not effectively substituted by tyrosine, phenylalanine or alanine, and it is invariant among members of family 2a, further demonstrating that this residue likely contributes to binding both through dehydration and by providing sorbent specificity via its hydrogen bonding potential. Tryptophans corresponding to W17 are also strictly conserved; the asparagine corresponding to N15 is also highly conserved (>60%), supporting the hypothesis that an intramolecular hydrogen bond between these residues is an important functional feature of family 2a modules. 4.2.3 C B M 2 a - C e l l u l o s e interaction observed by solid state N M R In biological applications, solid state N M R is an excellent tool for obtaining structural information for proteins that are not amenable to solution-based N M R techniques, or cannot be crystallized for X-crystallographic studies. Solid state N M R has been used to study prion proteins (49), membrane proteins (89), bacteriorhodopsins and gramicidin (110). The application of solid state N M R to the study of CBM-cel lulose interactions was tried in an attempt to directly collect structural information about C B M 2 a bound to the surface of insoluble cellulose. Ideally, specific information regarding protein amino acid residues involved in binding and the allomorphs of cellulose and which are interacting can be gleaned. This study is the first attempt to observe the interaction between a C B M and insoluble cellulose and it lays the foundation for further, more sophisticated, solid state N M R measurements. Since N M R relies upon observing nuclei that are have nuclear spin, cellulose, enriched in 1 3 C , and 1 5N-labelled C B M 2 a were produced. The C-4 carbon peaks of the 1 3 C - C P / M A S 110 spectrum are particularly indicative of cellulose structure, so it was decided that producing cellulose specifically labelled at C-4 would provide definitive information regarding the regions of the cellulose with which the C B M was interacting. The cellulose produced by A. xylinum cultures, however, was not uniquely labelled at C-4 (Figure 3.23); the bacterium, likely through a pentose phosphate pathway (30), scrambled a portion of the label. The signal to noise benefit for using 13C-enriched cellulose prompted the production of larger amount of labelled material using glycerol-1,3,- 1 3C 2 as a carbon source for the A. xylinum cultures resulting in cellulose that was had a significant proportion of 13C in positions 1, 3, 4, 6 of the glucose monomer component of cellulose. This cellulose was used to attempt the observation of CBM2a-cellulose interaction. Observation of CBM2a-cellulose interaction was attempted by a CP-drain experiment. This experiment is relatively robust and since it measures a decrease in the 1 3 C signals caused by only the interactions with the l 5 N nuclei, it should therefore unambiguously detect only surface binding of 1 5 N - C B M 2 a to 1 3C-enriched cellulose. The interaction between 1 5 N -C B M 2 a and 1 3C-enriched cellulose could not be directly observed by this experiment for a number of possible reasons. For example, 1 5 N - 1 3 C dipolar couplings are weak, effective to an upper limit distance of 4-5 A (118). 1 9 F - 1 3 C interactions however, are effective over a much greater distance. To take advantage of this fact, C B M 2 a has been produced, by a two-step expression method (101), that incorporates 5-fluoro-L-tryptophan. Immediate future study wil l focus on detecting the FW-CBM2a-cel lulose interaction. Since C B M 2 a moves on the cellulose surface (with a diffusion coefficient of 1.2 x 10"10 cm2-s"', (57)), any internuclear interaction wi l l be transient, only occurring for a fraction of the 10 ms CP-drain duration. The diffusion could be slowed by performing the experiments at a lower temperature and repeating the CP-drain experiment. I l l Some valuable information was obtained in this study; a 1 5 N - C P / M A S spectrum of i 5 N -C B M 2 a bound to the surface of cellulose was collected. A comparison of the , 5 N - C P / M A S spectrum with the projected 1 5 N spectrum of 1 5 N - C B M 2 in solution (Figure 3.25), shows that l 5 N - C B M 2 a bound to the surface is folded and has a conformation that is roughly similar to that in solution. The binding of C B M 2 a to the cellulose therefore, does not involve a significant conformational change, also supported by calorimetric evidence (C. A . Haynes, personal communication). With further refinement, solid state N M R should be successful in detecting and characterizing C B M interaction with insoluble cellulose. 4.3 Towards a model of CBM2a-cellulose Interaction 4.3.1 P r e v i o u s m o d e l s of b ind ing The specificity of C B M 2 a to bind crystalline cellulose and the identification of amino acid residues involved in binding provide a sound basis for formulating a putative binding model. Previous models for the binding of C B M s viewed crystalline cellulose as comprising layers of parallel cellulose molecules, with the sugar rings in successive layers perfectly superimposed. The surface to which the C B M binds would then be an ordered array of parallel cellulose chains, with the planes of the pyranose rings parallel to the surface and fully exposed to the solvent. Binding was postulated to occur by direct stacking of the exposed aromatic residues on the binding face of the C B M onto the exposed pyranose rings forming the (020) face of the cellulose crystal (77, 98, 130). The surface of crystalline cellulose, however, is more accurately described as a staircase (Figure 4.4) (51). In cross-section, viewed from one end, the crystal comprises layers with decreasing numbers of parallel cellulose molecules moving out to the vertices; the molecules in successive layers are off-set slightly with respect to those in the layer above it (5, 34, 45, 65). 112 4.3 .2 S u r f a c e a r e a of the e x p o s e d crysta l l ine f a c e s The largest solvent-exposed surfaces, the two (110) and the two (1-10) crystal faces, comprise mostly the edges of the sugar rings (Figure 4.4). In a perfect cellulose crystal, only the sugar rings of the apical cellulose molecules at two opposite vertices of the rectangular crystal have fully exposed faces. B M C C comprises bundles of microfibrils. Each microfibril presents two solvent-exposed crystalline, roughly rectangular, faces with cross-sectional dimensions estimated to be 15 nm ((110) face) by 40 nm ((1-10) face) (67, 136). Using the dimensions of the B M C C microfibril and the density of crystalline cellulose (1.5 g/cm 3) (81), we calculate the (110) crystal face to have a surface area of 3.34 x 105 cm 2 /g cellulose and the (1-10) face to have a surface area of 8.91 x 105 cm 2/g. As calculated from the solution structure, the maximum area shadowed by a bound C B M 2 a molecule is 1.32 x 10"13 cm 2 (calculated by D . L i m using the coordinates determined by X u et al. (141)). At maximum capacity, assuming monolayer surface coverage and confluent packing of the C B M on the sorbent surfaces, the (110) crystalline face could accommodate 4.2 u.moles C B M 2 a / g B M C C and the (1-10) face could accommodate 11.2 u,mole C B M 2 a / g B M C C . The predicted capacity of the two binding faces is therefore 15.4 u.moles/g. Given that the estimates of available cellulose surface area are based on a maximal fiber dispersal and monolayer surface coverage, the observed capacity of 11.7 u.moles/g cellulose (Table 3.1) agrees remarkably well with this estimated maximum value. The fully exposed cellulose chains, located at the two vertices of the rectangular fibril (the obtuse edges), could accommodate at most 0.91 u.mol/g cellulose, far less than the observed capacity. Therefore, C B M 2 a must be binding to one or both of the crystalline faces ((110) and (1-10)) of B M C C by interacting with the partially occluded sugar rings. The previous model based on stacking of fully exposed sugar rings leads to a predicted capacity far less than observed experimentally. 113 4.3 .3 A m o d e l for the b ind ing of C B M 2 a to crysta l l ine c e l l u l o s e Tryptophan residues are involved in many protein-carbohydrate interactions (95, 100, 122, 131, 132, 135). The non-covalent interactions between the aromatic residues of a protein and the sugar ring are not restricted to parallel stacking; there are likely many other conformations that are both thermodynamically stable and able to mediate ligand binding in crystalline cellulose. Since the great majority of pyranose rings of the glucose units are partially occluded in crystalline cellulose, the tryptophans can not stack directly on the sugar rings without a disruption of the cellulose structure. Other conformations, involving angled relationships or off-set parallel stacking that allow interaction between tryptophan and the sugars rings are therefore more likely to occur (78, 121, 135).The angle at which aromatic side chains are positioned with respect to the pyranose ring in protein-carbohydrate interactions varies from 17° to 52°; flat parallel ring stacking is not observed as frequently (135). With minimal movement from their equilibrium positions in the solution state, the surface tryptophans could form non-covalent interactions with the individual cellulose chains without substantial disruption of the crystal lattice. In C B M 2 a , the tryptophans essential for binding form a ridge along one face of the module. I propose that the tryptophans on this ridge bind to partially exposed cellulose chains, or 'steps', on the face of the cellulose crystal (Figure 4.4). The binding module tilts toward the 'staircase' so that other residues such as Q52 and N87, located alongside but slightly removed vertically from the vertex of the tryptophan ridge, are oriented in positions to potentially hydrogen bond or form van der Waal's interactions with the groups at the edges of the cellulose chains. The module may bind along one cellulose molecule, or straddle several 'steps' that comprise the cellulose surface. The arrangement of the tryptophan residues appears to allow contact with cellulose via only two pairs of the three tryptophans at once: either W17 and W54, or W54 and W72. This model is also supported by the ability of C B M 2 a to bind to B M C C when it has a bulky 114 carbohydrate moiety attached to N24, a residue adjacent to the row of tryptophans on the binding face (7). N24 is glycosylated when the polypeptide is produced in Pichia pastoris. C B M 2 a binds to the surface in such a way that the large, hydrophilic carbohydrate group is accomodated between the protein and cellulose surfaces. The binding of C B M 2 a to B M C C is dominated by entropic effects (25). Dehydration of the hydrophobic, tryptophan-rich protein surface and the crystalline cellulose surface drives binding. The specificity of the module for the cellulose surface, via the formation of hydrogen bonds and van der Waals contacts, is provided by both the surface tryptophans (especially W54) and other residues on the binding surface. The model proposed here agrees with this mechanism of binding. With the exception of N15, residues of C B M 2 a immediately adjacent to the tryptophan binding ridge that have hydrogen bonding potential have little overall effect on binding affinity. The binding of C B M 2 a to crystalline cellulose apparently irreversible, removal of C B M 2 a from solution does not result in desorption of C B M 2 a from the surface, yet binding is dynamic (12, 25, 88). The domain moves in two dimensions over the cellulose surface without ever fully dissociating from it (57). Sufficient multiple contacts must be maintained to prevent desorption. Since the module moves on the surface, the number of residues directly interacting with the cellulose surface must be in constant flux. For example, once C B M 2 a is bound to cellulose, none of the surface tryptophans is protected from oxidation by N-bromosuccinimide, although they are more difficult to oxidize (12). The accessibility to N -bromosuccinimide is consistent with a model for binding in which the tryptophans do not overlap the pyranose rings completely, and only two of the three conserved tryptophans bind to B M C C at any instant. The mutation W 5 4 A reduced affinity the most, suggesting that this residue plays the major role in binding. The N M R structure shows that W54 is not in the 115 same plane as W17 and W72; part of the importance of W54 could be its role in allowing pivoting between binding by W17/W54 and W54/W72 pairs. C B M 2 a could be envisioned to "waddle" across the cellulose, using W54 as the essential residue contacting the surface. 4.4 A general model for the binding of type A binding modules The abrogation or reduction of binding affinity when surface tryptophan residues of this and other C B M s of family 2a are modified by site-directed mutation or chemical modification clearly emphasizes the substantial role that tryptophans play in their binding to insoluble cellulose (12, 27, 93, 94). Additionally, the conservation of aromatic residues, such as tyrosine and phenylalanine, on an exposed surface is common in C B M s from families 1, 3, 5 and 10. Representatives from each of these families bind to crystalline cellulose and, as with C B M 2 a , mutation of the exposed aromatic residues reduces the affinity for cellulose (27, 85, 93, 94, 96, 97, 111). Many of the C B M 2 a variants constructed here with conservative substitutions maintain appreciable affinity for crystalline cellulose. It is likely that all of the type A C B M s , those binding binding modules with affinity for crystalline cellulose having a platform of exposed aromatic side chain residues, share a mode of binding where the surface aromatics drive binding and mediate contact with the staircase-like cellulose surface. 116 Figure 4 . 4 Three views each of two possible arrangements of C B M 2 a bound to the (110) face of crystalline celulose. I) C B M 2 a bound roughly along a single cellulose chain and II) C B M 2 a bound across a number of cellulose chains. (A) cross section of the microfibril, the internal cellulose chains are omitted for simplicity, (B) side view of the solvent-exposed surface and (C) view along the cellulose surface. The arrangements shown allows for at least two of the tryptophan residues (rendered as balls and sticks) instrumental in binding, to interact with the edge of the pyranose rings of the staircase-like surface of crystalline cellulose. 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