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Studies of noncovalent complexes of Cex and its inhibitors by mass spectrometry Tešić, Milica 2006

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STUDIES OF NONCOVALENT COMPLEXES OF CEX AND ITS INHIBITORS BY MASS SPECTROMETRY by MELICA TESIC B.Sc, Faculty of Physical Chemistry, University of Belgrade, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA December 2006 © Milica Tesic, 2006 Abstract Tandem mass spectrometry (MS/MS) has been used to compare gas phase and solution binding of three small-molecule inhibitors to the wild type and three mutant forms of the catalytic domain of Cex, an enzyme that hydrolyses xylan. The inhibitors, xylobiosyl-deoxynojirimycin, xylobiosyl-isofagomine lactam, and xylobiosyl-isofagomine consist of a common distal xylose linked to different proximal aza-sugars. The three mutant forms of the enzyme: Asn44Ala, Gln87Met and Gln87Tyr, alter the binding interactions between Cex and the distal sugar of each inhibitor. An electrospray ionization (ESI) triple quadrupole MS/MS system is used to measure the internal energies, A£ i n t , that must be added to gas phase ions to cause dissociation of the noncovalent enzyme-inhibitor complexes. Collision cross sections of ions of the apo-enzyme and enzyme-inhibitor complexes, which are required for the calculations of A E i n t , have also been measured. The results show that, in the gas phase, enzyme-inhibitor complexes have more compact, folded conformations than the corresponding apo-enzyme ions. With the mutant enzymes the effects of substituting a single residue can be detected. The energies required to dissociate the gas phase complexes follow the same trend as the values of AG0dissociation in solution. This trend is observed both with different inhibitors, which probe binding tothe proximal sugar, and with mutants of Cex, which probe binding to the distal sugar. Thus the gas phase complexes appear to retain much of their solution binding characteristics. Hydrogen/deuterium exchange shows that apo-protein ions from solutions of complexes exchange more hydrogens than ions of enzyme-inhibitor complexes from the same solution, indicating a more open conformation of apo-protein ions. Table of Contents Abstract ii Table of Contents iii List of Tables vi List of Figures vii List of Symbols and Abbreviations xii Acknowledgements xvii Dedication xviii C H A P T E R 1: Introduction 1 1.1. Principles of Electrospray Ionization Mass Spectrometry 2 1.2. Application of Mass Spectrometry to the Study of Noncovalent Complexes of Biomolecules 6 1.2.1. Biological Noncovalent Complexes 6 1.2.2. Biological Mass Spectrometry 7 1.2.3. Application of Mass Spectrometry to the Study of Noncovalent Complexes in Solution 8 1.2.4. Application of Mass Spectrometry to the Study of Noncovalent Complexes in the Gas Phase 10 1.2.5. Comparisons of the Solution and the Gas Phase Behaviour of Noncovalent Complexes 11 iii 1.3. Cex and its Inhibitors 12 1.4. Interaction Maps 17 1.5. Objectives of This Research 19 C H A P T E R 2: Experimental Methods 23 2.1. Instrumentation 23 2.2. Protein Preparation and Purification 29 2.3. Reagents, Materials and Sample Preparation 31 2.4. Calculation of Mass 31 2.5. Collision Cross Sections 32 2.6. Tandem Mass Spectrometry 34 2.7. Hydrogen/Deuterium Exchange 38 C H A P T E R 3: Mass Spectra of Cex and its Complexes 40 3.1. Spectra of Cex 40 3.2. Spectra of Complexes 43 3.3. Mass of Cex 46 C H A P T E R 4: Collision Cross Sections 48 4.1. Stopping Curves 48 4.2. Collision Cross Sections of WT Cex and Its Complexes 50 4.3. Collision Cross Sections of Mutants and Their Complexes 54 C H A P T E R 5: Binding Studies of Gas Phase Ions 59 5.1. MS/MS Spectra 59 5.2. Dissociation Curves 63 5.3. Dissociation at Different Collision Cell Pressures .....66 i v 5.4. Internal Energies and Reaction Times 69 5.5. Collision Cross Sections and Gas Phase Binding 74 5.6. Comparisons of Solution and Gas Phase Binding 76 5.6.1. Solution H/D Exchange 76 5.6.2. Comparison of AEint in the Gas Phase with AG°dissociation in Solution 84 CHAPTER 6: Conclusions and Recommendations for Future Work 87 References 90 v List of Tables Table 1.1 Inhibition constants, Kt, at pH 7 and 37 °C in 10 mM ammonium acetate. Values of Kt for the WT protein are taken from [82] and for the 20 mutants from [84] Table 4.1 Collision cross sections (A2) of WT Cex and WT complexes. The labels in the first row indicate the solution used, either protein alone, or protein and inhibitor 51 Table 4.2 Collision cross sections (A 2) of Gln87Met and Gln87Met complexes 54 Table 4.3 Collision cross sections (A 2) of Gln87Tyr and Gln87Tyr complexes 55 Table 4.4 Collision cross sections (A 2) of Asn44Ala and Asn44Ala complexes 55 Table 5.1 Free energy changes for the dissociation of complexes in solution and internal energies required for the dissociation of gas phase complexes in 25 us 73 Table 5.2 Total numbers of exchangeable hydrogens for WT Cex, Gln87Met, Gln87Tyr and Asn44Ala and their complexes with X 2 DNJ, X 2 IL and X 2 IF 77 Table 5.3 Maximum Ff/D exchange levels and saturation times for the +10 and +11 charge states of a) WT Cex, b) Gln87Met, c) Gln87Tyr and d) Asn44Ala and their complexes 80 vi List of Figures Figure 1.1 Electrospray interface and mechanisms of ion formation 5 Figure 1.2 Ribbon diagram of the three-dimensional {a/B)8 barrel fold of the catalytic domain of Cex (PDB Code: 2EXO) 1 3 Figure 1.3 Structures of xylobiose-derived inhibitors of Cex. The distal xylose is on the left, and the proximal sugar is on the right 16 Figure 1.4 Interaction map showing the binding between the active site of WT Cex and the proximal sugar and distal xylose of X 2 D N J (PDB Code: 1FH7) 18 Figure 2.1 Schematic of a quadrupole mass filter instrument 23 Figure 2.2 First stability region of the quadrupole mass filter 26 Figure 2.3 ESI-triple quadrupole mass spectrometer system. QO, RF only quadrupole (ion guide); Q0/Q1, Q1/Q2, Q2/Q3, ion lenses; SRO, small rods following QO; Q l , mass analyzing quadrupole; Q2, RF only quadrupole (collision cell); Q3, mass analyzing quadrupole; EXIT, exit aperture plate; C E M , detector; DEF, deflector 28 Figure 3.1 Mass spectrum of a solution of denatured WT Cex in 10% methanol and 1% acetic acid (pH 2.3) 41 Figure 3.2 Mass spectrum of a solution of native WT Cex in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8) 42 vii Figure 3.3 Mass spectrum of a solution of WT Cex and X 2 D N J in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8); [Enz] = 1 pM, [inh] = 29 uM, [Enz • Inh] = 4 pM 4 3 Figure 3.4 Mass spectrum of a solution of WT Cex and X 2 IL in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8); [Enz] = 0.04 pM, [inh] = 43 pM, [Enz • Inh] = 4.9 pM 4 4 Figure 3.5 Mass spectrum of a solution of WT Cex and X 2 IF in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8); [Enz] = 0.02 pM, [inh] = 34 pM, [Enz • Inh] = 4.9 pM 4 5 Figure 4.1 Stopping curves for the +10 charge state of WT Cex at different pressures of argon 49 Figure 4.2 E C-nhijln Linear plot of —In—— vs.—-—=— for the +10 charge of WT Cex. E m, The collision cross section a is determined by the slope of the line (the slope is 1.7783 according to the fit) 50 Figure 4.3 Collision cross sections vs. charge state for ions from a solution of WT Cex only and Enz and Enz • Inh ions from the solutions of the complexes of WT Cex with X 2 DNJ, X 2 IL and X 2 IF 52 Figure 4.4 Collision cross sections vs. charge state for native and denatured WT Cex 53 Figure 4.5 Collision cross sections vs. charge state for ions from the solutions of Gln87Met only and Enz and Enz • Inh ions from the solutions of viii its complexes 56 Figure 4.6 Collision cross sections vs. charge state for ions from the solutions of Gln87Tyr only and Enz and Enz • Inh ions from the solutions of its complexes 56 Figure 4.7 Collision cross sections vs. charge state for ions from the solutions of Asn44Ala only and Enz and Enz • Inh ions from the solutions of its complexes 57 Figure 4.8 Collision cross sections vs. charge state for ions from the solutions of proteins only 57 Figure 5.1 MS/MS spectra of + 10 WT Cex with X 2IF ions at a collision cell pressure of 1.5 mtorr, at ion initial kinetic energies of 150, 950, 1520, 1550, 1650 and 2150 eV 60 Figure 5.2 MS/MS spectra of + 11 WT Cex with X 2 DNJ ions at a collision cell pressure of 0.8 mtorr, at ion initial kinetic energies of 165, 605, 935, 1265, 1375 and 1485 eV 61 Figure 5.3 Relative abundances of the +10 Enz-lnh and +10 Enz ions in MS/MS spectra of WT Cex-X 2IF ions vs. Q0-Q2 rod offset voltage difference at a collision cell pressure of 1.5 mtorr of Ar 63 Figure 5.4 Relative abundances of the +77 EnzTnh, +11 Enz and +10 Enz ions in MS/MS spectra of WT Cex-X 2DNJ ions vs. Q0-Q2 rod offset voltage difference at a collision cell pressure of 0.8 mtorr of Ar 64 Figure 5.5 Relative abundances of the +77 Enz-lnh, and the sum of +77 Enz and +10 Enz ions in MS/MS spectra of WT Cex-X 2DNJ ions vs. ix Q0-Q2 rod offset voltage difference at a collision cell pressure of 0.8 mtorr of Ar 65 Figure 5.6 The dissociation voltages of the +10 and +11 charge states of the complexes of WT Cex with inhibitors X 2 DNJ, X 2 IL and X 2IF at different cell pressures 67 Figure 5.7 The dissociation voltages of the +10 and +11 charge states of the complexes of Gln87Met at different cell pressures 68 Figure 5.8 The dissociation voltages of the +10 and +11 charge states of the complexes of Gln87Tyr at different cell pressures 68 Figure 5.9 The dissociation voltages of the +10 and +11 charge states of the complexes of Asn44Ala at different cell pressures 69 Figure 5.10 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of WT Cex ^ Figure 5.11 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of Gin87Met 1^ Figure 5.12 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of Gln87Tyr ^2 Figure 5.13 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of x complexes of Asn44Ala 72 Figure 5.14 Collision cross sections of the complexes vs. the added internal energy to cause dissociation, A £ i n t , for a) +10 ions and b) +11 ions... 75 Figure 5.15 H/D exchange levels vs. time of the +10 and +11 charge states of Enz and Enz • Inh ions of the complex of WT Cex with X2IL 78 Figure 5.16 Maximum H/D exchange level vs. saturation time for a) +10 and b) + 11 charge states of Enz and Enz • Inh ions of all complexes 81 Figure 5.17 Maximum H/D exchange level vs. AG0dissodaliun in the solution for a) 83 +10 and b) +11 charge states of all the complexes Figure 5.18 Internal energies needed for dissociation of ions of the complexes of WT, Gln87Met, Gln87Tyr and Asn44Ala with inhibitors in the gas phase vs. A G 0 for dissociation of the complexes in solution for charge states a) +10 and b) +11 85 x i List of Symbols and Abbreviations Symbol or Abbreviation Description or Definition AC Ala APCI Asn Asn44Ala a u BIRD CD CEM CI CM COOH d DC DEF Projection area Alternating current Alanine Atmospheric pressure chemical ionization Asparagine Asparagine replaced by alanine at position 44 in the protein sequence Mathieu parameter Blackbody Infrared Radiative Dissociation Drag coefficient Channel electron multiplier Chemical ionization Centre of mass Carboxyl group Distance from the collision cell entrance Direct current Deflector Elemental charge Lab kinetic energy of an ion at the collision cell exit £ 0 Lab kinetic energy of an ion at the collision cell entrance ECM Centre of mass kinetic energy of an ion EDTA Ethylenediaminetetraacetic acid EI Electron ionization Enz Enzyme Enz • Inh Enzyme-inhibitor complex ESI Electrospray ionization EX1 H/D exchange mechanism 1 EX2 H/D exchange mechanism 2 / Frequency F Drag force FAB Fast atom bombardment FT-ICR Fourier Transform Ion Cyclotron Resonance Gin Glutamine Gln87Met Glutamine replaced by methionine at position 87 in the protein sequence Gln87Tyr Glutamine replaced by tyrosine at position 87 in the protein sequence Glu Glutamate (Glutamic acid) H/D Hydrogen/Deuterium His Histidine HIV Human immunodeficiency virus HSP Heat shock protein xiii ICP Inductively coupled plasma Inh Inhibitor k Boltzmann's constant K Inhibition constant I Collision cell length /' Collision cell length over which the reaction occurs LD Laser desorption Lys Lysine m Mass of an ion mA, mB, mc Ion masses mj Mass of the ion of interest m2 Collision gas mass M M=m,+m2 Mp Molecular mass of the protein MALDI Matrix assisted laser desorption ionization Met Methionine MS Mass spectrometry MS/MS Mass spectrometry/mass spectrometry M W Molecular weight m/z Mass-to-charge ratio n Gas number density TV Number of collisions N H 2 Amino group xiv OH Hydroxyl group P Pressure PDB Protein data bank QO RF only quadrupole (ion guide) Q0/Q1, Q1/Q2, Q2/Q3 Ion lenses Q l First quadrupole (mass analyzer) Q2 Second quadrupole (collision cell) Q3 Third quadrupole (mass analyzer) qu Mathieu parameter r0 Distance from the quadrupole centre to a rod RF Radiofrequency SH Thiol group SIMS Secondary ion mass spectrometry SRO Pre filter t Time T Temperature TI Thermal ionization t Reaction time r Trp Tryptophan Tyr Tyrosine u Coordinate x or y U DC voltage applied between opposite sets of rods xv v Ion speed near the collision cell exit vo Ion speed at the collision cell entrance V RF voltage applied between opposite sets of rods WT Wild type X j , x2 Mass to charge ratios X 2 DNJ Xylobiosyl-deoxynojirimycin X 2 IL Xylobiosyl-isofagomine lactam X 2 IF Xylobiosyl-isofagomine z Charge state AEint Added internal energy AQ0 Standard Gibbs free energy change A / / 0 Standard enthalpy change Az Traveling distance X Mean free path a Collision cross section 0 Average fraction of the centre of mass kinetic energy transferred to internal energy in a single collision co Angular frequency of the RF voltage £ x cot xvi Acknowledgements I would like to thank Don for teaching me how to approach science in the right way, how to be more efficient and for being a mass spec encyclopedia. I am very thankful to Aaron for teaching me stuff, Xian Zhen for the same and lots more, and Anthony, for being such a ray of light on the rainy day. Special thanks to Annie and her family, for becoming my friends for life. I thank the rest of the Douglas Lab 2001-2006 for chatsi beers, barbeques, group dinners, and lots of fun. I am especially thankful to Dave Bains and Dave Tonkin from electronics for always being on time and always nice and friendly. I would like to thank my dad for his support, friendship, sharing his wisdom with me, making me laugh and cheering me on along my life journey. To thank my mom or express gratitude are words not big enough. To my mom, who made me who and what I am today, for understanding every crazy thing that I would think of, for being my guide, my soul, my pillar of hope, and for not letting me quit: I just wish I will one day become at least a bit of the greatest human being you are. And last, but not least, I thank Kev, my other half. I had to cross the world to find him, but I did, and here I am, not being able to imagine going through the last five years without him. I thank him for always being there, making me laugh until it hurts, for putting up with me which is not always an easy thing to do. To Kev, my partner in crime and my best friend: Thank you for loving me. To all my friends in Vancouver and Belgrade: all this schooling would not be as half as fun if I did not have you. T H A N K Y O U ! xvii Mama, this is for you! xvin Chapter 1 Introduction Mass spectrometry is an analytical technique used to identify new compounds, quantify known compounds, study the structure and chemical, physical and biological properties of molecules, determine the isotopic composition of elements in compounds and study the fundamentals of gas phase ion chemistry. A typical mass spectrometric analysis consists of three stages: ionization, mass analysis and ion detection. Ionization methods in mass spectrometry are numerous: electrospray ionization (ESI), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), electron ionization (EI), matrix assisted laser desorption ionization (MALDI), thermal ionization (TI), secondary ion mass spectrometry (SIMS), fast atom bombardment (FAB), and inductively coupled plasma (ICP). There are many types of mass analyzers: sector, time-of-flight, quadrupole, quadrupole ion trap, Fourier transform ion cyclotron resonance and electrostatic ion trap. The choice of the ionization method and mass analyzer depends on the nature of the sample and the goal of the study. For the work presented in this thesis, an ESI triple-quadrupole mass spectrometer was used to investigate binding and other properties of noncovalent protein-ligand complexes in the gas phase. 1 1.1. Principles of Electrospray Ionization Mass Spectrometry Electrospray ionization was developed in the late nineteen sixties by Dole and coworkers [1-3]. In this early work, the simple experimental setup consisted of a spray chamber, nozzle-skimmer region, and a Faraday cage for ion detection. A liquid sample was electrosprayed through a syringe into the spray (evaporation) chamber. Drops evaporated in a flowing nitrogen gas at atmospheric pressure to form gaseous ions. Negative ions were investigated in these experiments. In the early nineteen eighties, John B. Fenn and coworkers first used electrospray as an ion source for a mass spectrometer [4-6]. At that time, ionization techniques that do not cause fragmentation of non-volatile analytes, named "soft", included very fast deposition of energy on a surface of a liquid or solid on which analyte is dispersed. These techniques for ionization of non-volatile analytes were based on impact of molecules of interest with ions in SIMS, atoms in FAB or photons in laser desorption (LD). Nevertheless, these ionization techniques produced very low product ion currents which decreased rapidly with the size of an ion. Other soft ionization techniques available at that time included CI and APCI. In CI, an analyte is ionized by ion-molecule reactions. APCI is a form of CI at atmospheric pressure. Mobile phase containing analyte is heated and then sprayed with high flow-rate nitrogen to form an aerosol. This aerosol evaporates to give neutral gas phase analyte molecules, which then undergo a CI process. These two techniques were used for the analyses of small molecules. Therefore, there was interest in developing a technique suitable for ionization of large biomolecules [7]. Many samples, including 2 biomolecules, are not sufficiently thermally stable to survive volatilization before ionization [8]. The soft mechanism of ESI allows for the formation of ions from these high molecular weight biomolecules without fragmentation. Besides ESI, MALDI is the most commonly used soft ionization techniques to study biomolecules by mass spectrometry. In MALDI, the analyte is dissolved in a solution of matrix material in organic solvents and water, after which it is spotted on a MALDI plate. After evaporation of the solvent, the analyte is ionized by a pulsed laser. During the electrospray process, ions are formed directly from solution. An analyte, which is ionized in a solution, is transferred into the gas phase at atmospheric pressure. ESI is considered a soft ionization technique since it produces intact ions, without significant fragmentation [8, 9]. As shown in Figure 1.1, an analyte solution is passed through a capillary (the "sprayer") which is kept at a high potential, usually 3-5 kV. The electric field at the tip of the capillary creates a fine spray of charged droplets; the droplets are attracted towards the mass spectrometer by the potential difference between the sprayer and a counter electrode. The mechanism of the electrospray process includes three stages: droplet formation, droplet shrinkage and gas phase ion formation [8]. In the positive ion mode, the solution at the tip of the electrospray capillary is subjected to a positive potential which forces positive ions from the solution to accumulate at the solution surface. This charging forces the solution to form an elongated meniscus, called a Taylor cone, in the downfield direction. As the solvent evaporates from the charged droplets, the charge density on the 3 droplet surfaces increases until the diameter of the drop reaches the Rayleigh limit [5, 10]. At this point the repulsion from the charges overcomes the surface tension holding the drop together and fission, or a Coulombic explosion, of an initial droplet into smaller droplets, occurs. The solvent evaporation is sometimes facilitated by the use of a nebulizer gas [11], usually compressed air. Two mechanisms for the creation of the gas phase ions from small charged droplets have been proposed and discussed in the last two decades [1-3, 12-14]. Figure 1.1 includes the two proposed mechanisms of gas phase ion formation. The first, "the charged residue" mechanism, was proposed by Dole and coworkers in 1968 [1-3], and later improved by Rollgen and coworkers [12]. According to this model, continuous reduction of the droplet size by solvent evaporation and fission ultimately results in the formation of such a small droplet that it contains only a single ion. The charge of the single ion comes from the residual charges, after solvent evaporation, that were accumulated on the droplet during the electrospray process [9]. The second, the "ion evaporation" mechanism, was first developed by Iribarne and Thomson in 1976 [13, 14]. Ions are believed to evaporate from the small highly charged droplets when the strong repulsion from the same-charge analyte ions, as well as from the charges on the solvent, overcomes the surface tension of the droplet. The ion evaporation model is considered to be the dominant mechanism for the production of small ions, mainly organic and inorganic ions, while the charged residue model applies to larger ions, such as proteins and their complexes [9]. 4 N 2 curtain gas Figure 1.1 Electrospray interface and mechanisms of ion formation. In an ESI mass spectrometer system, following their formation by ESI, ions pass into vacuum and then through a series of focusing ion lenses to reach a mass analyzer, which separates the ions according to their mass to charge ratios (m/z) and then to a detector that records the number of ions at each m/z value. From formation to detection, ions pass through several regions of decreasing pressure. Typically, differential pumping is used to increase the ion sampling efficiency. After formation at atmospheric pressure, ions enter 5 a region between a curtain plate and orifice plate, which contains a counter flow of curtain gas, usually nitrogen, at atmospheric pressure. The curtain gas is used to further assist droplet evaporation and to break down ion clusters and remove residual solvent. After passing through an orifice, ions enter the first vacuum stage at a pressure of about 2 torr. Ions are then sampled by a skimmer as they enter the next vacuum stage, usually containing an ion guide, that transports and focusses the ions toward a mass analyzer. This stage has a lower pressure, in the range of mtorr. The quadrupole mass analyzer and the detector are situated in a final vacuum stage, which usually has a pressure of about 10"5-10 6 torr. 1.2. Application of Mass Spectrometry to the Study of Noncovalent Complexes of Biomolecules 1.2.1. Biological Noncovalent Complexes Biological complexes containing units that are held together by ionic, van der Waals or hydrophobic interactions, or hydrogen bonds, are termed noncovalent. Noncovalent complexes of proteins with other proteins, various ligands, metal ions, peptides and oligonucleotides play a crucial role in the biological functions of proteins, such as cellular function, growth and differentiation, bacterial and viral infections, inflammation and immune response. The disruption of these interactions leads to disease and death of a cell [15, 16]. Proteins, in complexes with themselves.or other proteins, form oligomers that have either functional roles or are involved in signalling pathways. Studying these 6 oligomers gives insights into enzymatic regulation. It also provides information on protein stability, since protein assemblies increase the stability of constituent proteins and prevent thermal degradation [9]. Noncovalent protein-ligand complexes are of great importance in drug discovery and cancer research. Inhibitors from both synthetic and natural sources bind noncovalently to disease-causing proteins disrupting the mechanism of the disease [15]. Metal ions are a crucial factor in the catalytic activity of many enzymes. Diseases like arthritis and cancer arise partly from the malfunction of metalloproteinase enzymes, which require zinc and calcium for activity [16]. Peptides can act as protein inhibitors, through a variety of mechanisms [17]. For example, after binding to a protein, a peptide can cause conformational changes of an entire protein or some of the domains on a protein. Genetic information in nucleic acids is regulated by proteins, which bind to nucleic acids by highly specific noncovalent interactions. Many significant biological functions like DNA replication, transcription and translation are controlled by protein-DNA interactions [17]. There is interest in developing new techniques to monitor all these types of interactions. Better understanding of the structures of all these types of complexes and their internal interactions can provide insights into their mechanisms of action. 1.2.2. Biological Mass Spectrometry In the nineteen eighties, ESI and MALDI, were first employed to study proteins [7, 18]. With the development of these soft ionization methods, mass spectrometry has become an 7 important technology in life sciences research, supplying information on molecular masses and primary sequences of proteins. Furthermore, ESI and MALDI mass spectrometers are used in structural biology for studies of secondary, tertiary and quaternary structures of proteins. Noncovalent complexes of proteins were first observed by ESI mass spectrometry in 1991 [19]. Since then, they have been extensively studied. In 2002, the Nobel Prize in chemistry was awarded "for the development of methods for identification and structure analyses of biological macromolecules". John B. Fenn and Koichi Tanaka, the pioneers in the utilization of the two ground breaking soft ionization techniques to study biological macromolecules, ESI and MALDI, shared half of the prize. The second half of the Nobel Prize in the same year was awarded to Kurt Wtithrich for development of nuclear magnetic resonance (NMR) spectroscopy for determining the three-dimensional structure of biological macromolecules in solution. 1.2.3. Application of Mass Spectrometry to the Study of Noncovalent Complexes in Solution In the last fifteen years mass spectrometry has been used to study proteins [20-23] and their noncovalent complexes in solution [9, 15, 16]. Both positive and negative ion mode ESI-MS have been applied for these studies. Mass spectrometry has been used to determine the conformations and quaternary structures of proteins and their complexes, previously characterized by other methods. ESI-MS has been used to determine the stoichiometry of known and unknown protein complexes in solution, since the molecular 8 mass of oligomers directly shows the number of subunits in the quaternary structure [9, 24-27]. Allosteric interactions between complex constituents in some cases lead to cooperative binding of substrates and ligands. The stability of the complexes and of the binding of the components of the complex can be probed by mass spectrometry [28, 29]. Environmental factors, like pH and temperature, have a great influence on the conformation of proteins and the dynamics of protein complexes. By coupling temperature controlling elements to an electrospray source [30] or by controlling the temperature of the solution [31], it is possible to probe the thermal unfolding of protein complexes. Many types of solution protein-ligand complexes have been studied by ESI-MS: antibody-antigen, protein-cofactor, enzyme-substrate etc. Their dissociation constants [32], and formation of complexes or intermediates [33-36] have been studied in detail. Protein-metal ion complexes have been studied by MS. The most common application is the determination of the metal ion-binding stoichiometry [37]. Protein-peptide complexes have been studied extensively by ESI-MS [16, 17] including screening of peptide libraries for identification of inhibitors [38] and determination of the protein conformation upon peptide binding [39], the probing of structural dynamics by hydrogen/deuterium (H/D) exchange [40], and determination of structural and kinetic properties of a protein-peptide interactions [41]. Protein-RNA recognition in solution has been often studied by ESI-MS [42, 43]. This recognition is important in the replication cycle of HIV and in the discovery of targeted inhibitors of protein-RNA complexes [44]. Protein-DNA binding affinity can be determined by ESI-MS [45]. 9 1.2.4. Application of JVIass Spectrometry to the Study of Noncovalent Complexes in the Gas Phase The study of gas phase noncovalent complexes may lead to conclusions about the role of solvent in the binding. In addition, comparison of solution and gas phase binding can give an indication of the possible use of mass spectrometry for fast and highly sensitive determination of conformation, stoichiometry and dissociation energetics of these complexes. Since the gas phase ions can be completely free of solvent, the ensemble of conformations adopted by a gas phase protein may differ from that of the same protein in solution [46], leading to differences in the structural and energetic bases for noncovalent interactions of proteins with other entities. In some cases, water molecules are directly involved in the solution binding interactions [47], and therefore in a gas phase complex free of water, additional differences in binding may occur. Nevertheless, there have been several reports of complexes that retain at least some of their solution binding characteristics [48-57]. Various mass spectrometric techniques are used to study biomolecules in the gas phase, in order to obtain details about topology (sequence of secondary structure elements such as a helices and [3 sheets), quaternary structure and stability [9]. These techniques include blackbody infrared radiative dissociation (BIRD) [58, 59], dissociation in the orifice-10 skimmer region of an ion sampling interface [48], measurements of H/D exchange of trapped ions [60, 61], tandem mass spectrometry (MS/MS) [62], and collision cross sections measured by ion energy loss [63] or ion mobility [64] experiments. An experimentally tractable method to investigate the details of binding in gas phase complexes is to make systematic changes to the binding partners. This method has been used to investigate the effects of individual hydrogen bonds between heme and myoglobin and cytochrome bs [48], binding in double stranded DNA [49, 50], as well as to investigate binding of inhibitors to aldose reductase [51], peptides to vancomycin [52] and avoparcin [53], small molecules to carbonic anhydrase [54], peptides to OppA [55], an antibody single chain fragment to a series of structurally related trisaccharides [47, 56], and a series of trisaccharides to an antibody single chain fragment [57]. 1.2.5. Comparison of the Solution and the Gas Phase Behaviour of Noncovalent Complexes Mass spectrometry has proved to be a fast, sensitive, and highly specific method for studying conformations and binding of biomolecules and their complexes. However, it is necessary to determine how gas phase binding data correlate to solution data in order to make MS data of value. In some cases, there is no correlation between solution and gas phase binding. Examples include various decomposition studies: tetrameric assemblies of avidin [65], dodecamers of the small heat shock protein HSP16.9 [30], as well as hemoglobin tetramers [26] and the hexameric chaperone complex MtGimC [66]. A 11 common dissociation channel for all these complexes in the gas phase is loss of a monomer or a single small unit. This differs from the solution dissociation, where the products are oligomers. On the other hand, many studies report a correlation between solution and gas phase properties. Complementary DNA duplexes in the gas phase were found to be more stable than non-complementary duplexes, which correlated to solution binding and showed that specific hydrogen bonding interactions between DNA strands prevail even in the absence of water molecules [49]. It has also been found that relative energies for dissociation of heme from a series of mutants of myoglobin and cytochrome bs in the gas phase increase with the increase in the activation energies for heme loss in solution [48]. It is still not known in what cases gas phase complexes will show solution binding properties, although it is argued that the type of the noncovalent interaction, the structure and strength of the gas phase complex and the control of instrumental variables require investigation [16]. 1.3. Cex and its Inhibitors This thesis describes a study of the gas phase complexes between the enzyme Cex and a 12 series of three disaccharide inhibitors. There are about 10 possible isomers of small oligosaccharides, and this provides for an enormous structural and functional diversity [67]. Oligo- and polysaccharides play a central role in many biological processes such as food storage and utilization, viral infection and highly specific cell signalling processes [68, 69]. Selective hydrolysis of glycosidic bonds between two carbohydrates or a carbohydrate and a non-carbohydrate is very important for the utilization of energy, 12 breakdown and expansion of cell walls and proper functioning of signalling molecules. The enzymes hydrolysing these bonds are called glycosyl hydrolases. Mechanisms of their action have been extensively studied [70-75]. Glycosyl hydrolases are classified into families based on similarities of their amino acid sequences [68]. Figure 1.2 Ribbon diagram of the three-dimensional {a/0)s barrel fold of the catalytic domain of Cex (PDB Code: 2EXO). Cex (cellulasexylanase) is a member of the family 10 glycosyl hydrolase found in the soil microorganism Cellulomonas fimi. It consists of a C-terminal cellulose binding domain, and an N-terminal catalytic domain which is involved in substrate and inhibitor binding. These two domains are connected by a polypeptide containing proline and threonine residues [76]. Catalytic domain of Cex, which was used in this study, folds into (a/ R)8 13 barrel. Its three-dimensional structure is shown in Figure 1.2. Cex catalyzes the hydrolytic cleavage of xylan (0-1, 4-linked polymers of xylose), and is therefore categorized as a xylanase [77]. Xylan, together with cellulose, is a major component of grass-like plants and hardwoods [78]. Applications of Cex are widespread with the most important including fuel production and biomass conversion [79]. In order to investigate in detail the role of the key residues involved in the catalytic activity of Cex, methods such as amino acid isotopic labelling, crystallography or kinetic studies of mutants have been used. Isotopic labelling involves the placement of labelled 2 13 15 amino acids (containing isotopes like H, C, N etc.) instead of the ordinary amino acids suspected to be involved in binding of substrates, and the detection of the position of these labelled amino acids in the complex by NMR. Crystallography is a method for determining the arrangement of atoms in crystals. Crystals are irradiated by x-rays and angles of diffraction are measured and used to determine the distances between atoms and to create electron density maps. This directly provides the three-dimensional structure of the complexes. Kinetic studies of mutants involve systematic mutation of amino acids thought to be involved in binding at the active site, and measurement of the consequent enzyme activity [78, 80-82]. Activities of mutant enzymes for hydrolysis of substrates are significantly lowered compared to the wild type (WT) enzyme. The importance of individual interactions between the enzyme and substrate for the catalysis is determined by comparison of the kinetic parameters of the mutant compared to the WT protein [78]. 14 Five high-affinity xylobiose-derived azasugars that inhibit the catalytic activity of Cex have been described: imidazole, lactam oxime, deoxynojirimycin, isofagomine lactam and isofagomine [82]. They all exhibit competitive inhibition, i. e. the formation of an enzyme-inhibitor complex noncovalently bound at the active site. Three-dimensional structures of complexes of each inhibitor with Cex have been determined [83]. Solution binding affinities for these inhibitors have been determined [82, 84] as inhibition constants, Kt, which represent dissociation constants for the following equilibrium [82]: Enz + Inh Enz-Inh (1.1) with an inhibition constant: (L2) [Enz • Inh\ where Enz represents the enzyme, Inh represents an inhibitor and Enz • Inh represents the complex. Figure 1.3 shows the structures of the three inhibitors used in this study, with binding affinity increasing in the order: xylobiosyl-deoxynojirimycin (X 2DNJ), xylobiosyl-isofagomine lactam (X2IL) and xylobiosyl-isofagomine (X2IF). All inhibitors consist of two sugars: a common distal xylose, and a distinct proximal aza-sugar [77, 83]. Cex binds 15 to the proximal and distal sugars through van der Waals interactions, electrostatic interactions and a series of hydrogen bonds to the amino acid side chains. The distal xylose from the inhibitor binds to a series of amino acids in the Cex sequence called the OH OH Xylobiosyl Deoxynojirimycin ( X 2 D N J ) M W = 265 Da OH Xylobiosyl Isofagomine ( X 2 I F ) M W = 249 D a Figure 1.3 Structures of xylobiose-derived inhibitors of Cex. The distal xylose is on the left, and the proximal sugar is on the right. -2 sub-site, while the proximal sugar from the inhibitor binds to the another series of amino acids in Cex, named the -1 sub-site, as described below in detail. Since all the inhibitors have the distal xylose in common, changes in the inhibitor probe binding of a 16 given form of Cex to the proximal sugar. As all the mutations that were used in this study alter the amino acids that bind to the distal xylose, for the same inhibitor, the mutant proteins probe binding to this distal sugar. 1.4. Interaction Maps The x-ray crystal structures of the complexes provide insights into details of the binding of the complexes [82, 83]. Using x-ray structural information and PDB Viewer software [85], it is possible to create a two-dimensional map of hydrogen bonds between the proteins and inhibitors. Figure 1.4 shows, for example, the hydrogen bonds between the active site of WT Cex and X 2 DNJ [83]. The distal xylose from X 2 DNJ binds to the -2 sub-site of Cex by forming hydrogen bonds with the Lys47, Gln87, Asn44, Glu43 and Trp273 residues. The proximal sugar binds to the -1 sub-site of Cex by forming hydrogen bonds with the Lys47, His80, Asnl26, Glu233, and Gln203 residues [77, 83]. In comparison, when WT. Cex binds X 2 IF [83] the interactions with the distal xylose are largely preserved, although the hydrogen bonds may have different lengths. At the proximal sugar, only the bond between His80 and OH-3 is preserved, but with a different length. Two new bonds are formed with the "anomeric" nitrogen N- l : one with the nucleophile Glu233 and a second with Gln203. When WT Cex binds X 2 IL [82, 84] instead of X 2 DNJ, again the hydrogen bonds with the distal xylose are largely preserved. At the proximal sugar, only the interaction between His80 and OH-3 is preserved. There are new interactions of 0-2 with His80, Asnl26 and the catalytic nucleophile Glu233, 17 and of nitrogen N - l with the acid-base catalyst Glul27 and the catalytic nucleophile Gln203. Asn44 Gln203 Lys47 His80 v ; v > -2 sub-site -1 sub-site Figure 1.4 Interaction map showing the binding between the active site of WT Cex and the proximal sugar and distal xylose of X 2 DNJ (PDB Code: 1FH7). 1.5. Objectives of This Research Cex is well characterized in crystal state and in solution by x-ray crystallography, NMR and kinetic studies of mutants, and therefore provides a good opportunity to study gas phase binding in detail. Cex forms numerous covalent and noncovalent complexes as a WT or a mutant protein with a series of inhibitors. These complexes have a range of 18 binding strengths. It is especially attractive to investigate the potential of mass spectrometry to distinguish small differences in binding energies. Mass spectrometry, if proven to show the same trend in binding energetics in gas phase complexes as in solution complexes, can be used at least as a preliminary method for qualitatively determining binding energetics and in some cases stoichiometry. This is important since mass spectrometry has advantages of speed and sensitivity over other analytical methods. In this study, collision cross section measurements, tandem mass spectrometry and hydrogen/deuterium exchange are used to characterize, in detail, the binding energetics and conformations of noncovalent complexes of the catalytic domain of the WT and three mutant forms of the 0-1,4 glucanase Cex, with three disaccharide inhibitors in the gas phase, with comparisons to solution properties. Among numerous mutant forms of Cex available for analysis, three were selected for this study, due to the wide range of binding strengths of their complexes with the inhibitors: Gln87Met, Gln87Tyr, and Asn44Ala. Values of Kt for the complexes used in this study are shown in Table 1.1. Probing of individual hydrogen bonds between the enzyme and inhibitors was possible with this set of noncovalent complexes. Based upon the crystal structures of WT Cex with each of the inhibitors [83], as well as the structures of some of the mutants with other inhibitors [81], it is possible to predict the consequences of mutations upon ligand binding. In general, with mutant proteins, the interactions, with the proximal sugar will be preserved as they are in WT Cex [81]. With the Asn44Ala mutant, the interaction between Asn44 and OH-5 of the distal xylose is eliminated for all three 19 inhibitors. Likewise, with the Gln87Met mutant, the hydrogen bond between Gln87 and 0-2 of the distal xylose is removed for all three inhibitors. However, with the Gln87Tyr mutant, while the bond between Gln87 and 0-2 of the distal xylose is removed, a new bond is formed with OH-4. tf,(uM) X 2 DNJ X 2 IL X 2JP WT 5.8 0.34 0.13 Gln87Met 18 2.1 0.39 Gln87Tyr 62 11 1.2 Asn44Ala 180 24 7.1 Table 1.1 Inhibition constants, Kt, at pH 7 and 37 °C in 10 mM ammonium acetate. Values of Kt for the WT protein are taken from [82] and for the mutants from [84]. In this study, tandem mass spectrometry was used to determine the internal energies, AEim , that must be added to gas phase ions of the complexes to cause dissociation of the complexes. Collision cross sections, which are required to calculate AE i n t , were measured by ion energy loss experiments. Different dissociation mechanisms are observed for different complexes. For WT Cex, the complex with the strongest inhibitor, X 2IF, exhibits only neutral inhibitor loss, while the complexes with the weaker inhibitors X 2 DNJ and X 2 IL show both neutral and some (< 20%) charged inhibitor loss. The ratio of charged-to-neutral inhibitor loss is largely independent of collision energy. All mutant proteins show only neutral inhibitor losses. 20 In gas phase ions, binding of the inhibitors appears to contribute significantly to the stabilization and the folding of the protein, since complexes that are more strongly bound have lower collision cross sections. Solution H/D exchange experiments show that ions of the complexes have more compact conformations than protein ions formed from the same solutions. Values of AZsint were compared to change in standard Gibbs free energy A G 0 for dissociation of the complexes in solution. The results show that the complexes that are more strongly bound in solution require higher internal energies to cause dissociation in the gas phase. This trend was observed both with different inhibitors and with the mutant proteins. Thus binding of the enzyme to both the proximal and distal sugars in the gas phase seems to retain many of its solution properties. Some of the work described in this thesis is published in the Journal of the American Society for Mass Spectrometry as: ' Milica Tesic, Jacqueline Wicki, David K. Y. Poon, Stephen G. Withers, Donald J. Douglas, Gas Phase Noncovalent Protein Complexes that Retain Solution Binding Properties: Binding of Xylobiose Inhibitors to the /J - l , 4 Exoglucanase from Cellulomonas fimi, J. Am. Soc. Mass Spectrom., 2007,18, in press. Most of the chapters are greatly expanded, in comparison to the published article. Chapter 2 contains a more detailed description of instrument operation, protein and sample preparation, and models for calculations of collision cross sections and added internal 21 energies for dissociation of complexes. Chapter 3 contains detailed descriptions of mass spectra of Cex and its complexes, and a discussion of the conformations of native and denatured protein. Chapter 4 contains the results and a detailed discussion of the collision cross sections and conformations of Cex and its complexes. Chapter 5 includes the results of tandem mass spectrometry of gas phase complexes of Cex. The detailed discussion on the differences in the added internal energies to cause the dissociation of the complexes with different inhibitors or forms of the protein is provided. Chapter 5 also contains the results of solution H/D exchange experiments. The final chapter, Chapter 6, is a summary of this thesis with concluding remarks and recommendations for future work concerning related projects. 22 Chapter 2 Experimental Methods 2.1. Instrumentation The main components of a quadrupole mass spectrometer system are an ion source, ion lenses, quadrupole mass filter and a detector, shown schematically in Figure 2.1. Figure 2.1 Schematic of a quadrupole mass filter instrument. 23 A quadrupole mass filter consists of four parallel rods, arranged in a square configuration [86]. Hyperbolic rods would provide an ideal electrode geometry, but in practice, because of their ease of production, round rods with an optimized spacing are utilized [87]. Each pair of opposite rods in the x and y directions is connected and radiofrequency (RF) and direct current (DC) potentials are applied to create electric fields within the quadrupole [88]. The motion of an ion in a quadrupole field is described mathematically by solutions to a second-order linear differential equation, originally developed by Mathieu in 1868 [89]. Ions are injected into the quadrupole in the z direction, and their transverse motion in the xz and yz planes is described by the Mathieu equation: d2u + {au-2qucos2Z)u=0 (2.1) where u is one of the coordinates (x or y ), , » is the angular frequency of the R F voltage in rad s"1 (co = 2nf , where / is the frequency in Hz). The parameters au and qu are defined such that [86]: 4eU a u = a x = - a y = TT (2-2) mco r0 2eV <iu=qx=-qy=—TT ( 2 - 3 ) mco r0 24 where e is elemental charge, U is a constant time-dependent DC voltage applied between opposite sets of rods, V i s a time-varying RF voltage applied between opposite sets of rods, m is the mass of an ion, and r0 is the distance from the quadrupole centre to any rod surface. The solutions of equation (2.1) have been described in detail [90]. They describe trajectories of the ions in the quadrupole field. There are two types of solutions to this equation. "Unstable" trajectories have amplitudes of motion that tend toward infinite displacement from the centre of the quadrupole. As a result, ions are lost by collision with the quadrupole rods. Ions with "stable" trajectories, which represent the second type of solution to the Mathieu equation, are successfully transmitted through the analyzer and recorded by the detector [88]. Stability in both thex and y directions depends on the au and qu values. Combinations of au and qu giving stable solutions to the Mathieu equation can be plotted using au and qu as coordinates. By displaying these values for the* and y directions, a "stability diagram" for each direction is obtained. The overall stability diagram for the mass filter for motion in two dimensions can be obtained by superimposing the individual diagrams for the x and y directions. Figure 2.2 shows the stability region formed at one of intersections of the plots for the x and y directions. This stability region is of the most practical interest. Other stability regions require higher applied potentials, although they have been investigated for ICP-MS [91-94]. 25 scan line 0.2 — 0.1 —\ q = 0.908 0.2 0.4 0.6 0.8 Figure 2.2 First stability region of the quadrupole mass filter. a 2U Given that —=—- does not depend on m/z for a given U and V , masses of all ions q V are situated on the same line in the stability diagram, called a scan line, for which —=const. As the values of U and V change, so do the masses which are stable within the mass filter. For example, in Figure 2.2, the ion with the mass mB will be transmitted to the detector, since it is situated within the stability diagram. Ions with masses mA and m c will not be transmitted, since they lie outside the stability region [95]. Increasing the slope of the scan line brings it to the tip of the stability diagram, which increases the resolution and decreases the sensitivity [88]. If a DC voltage is not applied to the rods then ax =- ay =0, and the scan line is horizontal on the qx and - qy axis. In this case, the quadrupole operates in RF only mode and serves as an ion guide. 26 Experiments were performed with an in-house made ESI-triple quadrupole mass spectrometer system, which is schematically shown in Figure 2.3. The sprayer consisted of a 2 cm length of fused silica capillary (Polymicro Technologies, Phoenix, AZ), with inner and outer diameters of 76 and 150 um, respectively, connected to a 3 cm long stainless steel tube (Small Parts Inc., Miami Lakes, FL) with inner and outer diameters of 0.02 and 0.04 cm, respectively. Samples were infused to the stainless steel tube at 1 pL/min. High voltage (4000 V) was applied to the stainless steel tube. Ions pass through a nitrogen curtain gas (manufacturer's stated purity 99.999%, Praxair, Mississauga, ON), a 0.25 mm-diameter sampling orifice into a region with a background pressure of 1.3 torr, then through a skimmer with a 0.75 mm diameter orifice, into an RF only quadrupole ion guide, Q0, operated at 4xl0"3 torr. Ions then pass into the main chamber containing quadrupoles Q l , Q2 and Q3 (background pressure 8xl0"6 torr). A channel electron multiplier (CEM) or "channeltron", with ion counting was used for detection. A C E M is a horn-shaped continuous dynode structure that is coated on the inside with an electron emissive material, such as metal or PbO. An ion striking the C E M surface creates secondary electrons that have an avalanche effect to create more electrons and finally to produce a current pulse at the output. 27 ESI source. curtain' plate A collision gas QO orifice skimmer SRO Q l • • Q0/Q1 Q2 EXIT Q3 CEM Q1/Q2 Q2/Q3 T DEF Figure 2.3 ESI-triple quadrupole mass spectrometer system. QO, RF only quadrupole (ion guide); Q0/Q1, Q1/Q2, Q2/Q3, ion lenses; SRO, small rods following QO; Q l , mass analyzing quadrupole; Q2, RF only quadrupole (collision cell); Q3, mass analyzing quadrupole; EXIT, exit aperture plate; CEM, detector; DEF, deflector. The potential difference between the orifice and the skimmer was 100 V in all experiments. The curtain plate was held at 1400 V. The voltages on the lenses between the quadrupoles, Q0/Q1, Q1/Q2, Q2/Q3, and on the short RF only rods before Ql were 100, 103, 85, 103 V respectively. The aperture plate at the exit of Q3 was 0 V. For MS experiments the rod offsets on the quadrupoles Q0, Ql , Q2 and Q3 were 119 V, 110 V, 100 V and 90 V, respectively. For MS/MS experiments, the Q2 and Q3 rod offsets were varied, as described below. The frequency of the quadrupole RF was 0.768 MHz, giving a mass range of up to m/z = 6500 (quadrupole field radius, 4.16 mm). In all experiments the collision gas was argon (manufacturer's stated purity 99.9995%, Praxair, Mississauga, ON). The pressure in the collision cell (length 20.6 cm) was varied in the experiments and monitored by a capacitance manometer (MKS Instruments, Baratron 28 model 120AA, Boulder, CO). All experiments were repeated four times. Uncertainties are the standard deviations of four repeated experiments. 2.2 Protein Preparation and Purification WT Cex and its mutants were identically produced starting with a 20 mL overnight culture of an E. coli plasmid containing the Cex gene, in a synthetic medium containing ampicillin. A 10 mL sample of the resultant culture was used to inoculate 1 L of synthetic medium, grown to an optical density of -0.5 at 600 nm, induced with 0.1 mM isopropyl thiogalactoside, and harvested 48 hours after induction. The temperature was 30 °C throughout the entire growth and expression period. The cells were harvested by centrifugation (6,000 rpm, 20 min), re-suspended in -25 mL of 50 mM potassium phosphate, 0.02 % NaN 3 at pH 7.2 in H 2 0 (K-P-7 buffer), and lysed by passing twice through a French-press cell at 10,000 psi. After addition of Complete protease inhibitor cocktail (Roche Applied Science, Laval, QC), cell debris was removed by centrifugation (15,000 rpm for 30 min), and the supernatant, along with the original media supernatant, was stirred overnight at 4 °C with -20 g of degassed long fibrous cellulose (Sigma, Oakville, ON) suspended in K-P-7 buffer. The cellulose was packed into a -150 mL fast protein liquid chromatography column, washed with -270 mL of 1 M NaCl in K-P-7 buffer, and followed by -200 mL of K-P-7 buffer. The protein was eluted with FL-O at 1 mL/min while collecting 10 mL fractions. The appropriate fractions were pooled and concentrated to -10 mL using a 10 kDa molecular weight cut-off stirred 29 ultrafiltration cell (Amicon Corp., Danvers, MA), and exchanged into a buffer containing 20 mM sodium phosphate, 10 mM EDTA, and 20 mM cysteine hydrochloride at pH 7. Washed agarose-immobilized papain (150 pL) (Pierce, Rockford, IL) was then added to cleave Cex at multiple sites within the linker region between its catalytic and cellulose binding domains. After incubation overnight at 37 °C on a tube roller, the papain was removed by centrifugation at 5,000 rpm. The agarose beads were rinsed three times with K-P-7 buffer, and all the supernatants were combined and incubated overnight at 4 °C on a tube roller with 2 g of washed avicel (Fluka Biochemika, Buchs, Switzerland). The avicel, with bound uncleaved Cex and the isolated cellulose binding domain, was removed by centrifugation at 5,000 rpm. The supernatant, containing the catalytic domain of Cex, was concentrated by ultrafiltration using a 3 kDa molecular weight cut-off membrane and exchanged into 20 mM potassium phosphate (K-P-8) buffer (pH 8), loaded onto a Hi-Trap Q HP column (GE Healthcare Life Sciences, Piscataway, NJ), and washed with 10 mL of 20 mM K-P-8 buffer (pH 8). The various mutants were eluted from the column using the above washing buffer supplemented with 1.0 M NaCl at a flow rate of 0.5 mL/min. Proteins can be detected from 0.6 M to 1.0 M NaCl, and the appropriate fractions were collected, buffer-exchanged into distilled H 2 O , and concentrated to a final volume of ~1 mL. The final enzyme concentrations were determined using a U V spectrophotometer with a predicted value [96] of molar extinction coefficient e2 8onm = 52,870 M'cm" 1 [97]. 30 2.3 Reagents, Materials and Sample Preparation Solutions of WT Cex or its mutants were 10 uM. Solutions of the complexes were prepared by mixing the enzyme and the inhibitors in ratios calculated to produce nearly fully complexed enzyme in solution. For example, for the W T - X 2 D N J complex, 17 pL of a 2.4 mg/mL (69 pM) stock solution of WT Cex was mixed with 2 pL of a 4.0 mM stock solution of X 2 DNJ in a total volume of 240 pL. The resulting equilibrium concentrations of the enzyme, inhibitor and complex, calculated from the Ki value, are about 1, 29, and 4 pM, respectively. Solutions contained 10% methanol (HPLC grade, Fisher Scientific, Fair Lawn, NJ), and 10 mM ammonium acetate (Fisher Scientific, Fair Lawn, NJ) buffer, to minimize pH changes in electrospray. The addition of 10% alcohol to a protein solution does not generally lead to conformational changes, unless the protein is otherwise near to a folding transition [98, 99]. Solution pH was measured with an Accumet pH meter (model 15, Fisher Scientific, Arvada, CO). Solutions of the complexes with mutants were prepared in the same manner. For H/D exchange experiments, deuterium oxide D 2 0 (99.9% D) and methanol-d4 (99.8% D) (Cambridge Isotope Laboratories Inc., Andover, MA) were used. 2.4 Calculation of Mass Mass spectra were processed using Bio Multiview software (Sciex, Thornhill, ON) to determine the molecular mass of Cex by deconvolution of the multiply charged ions of Cex. Peaks were selected either automatically by the computer, or manually. The average 31 from six measurements of mass was used as a final value for the mass of Cex. The uncertainty in the mass is the standard deviation of these six measurements. 2.5 Collision Cross Sections Collision cross sections give a measure of the size of an ion. For proteins and their complexes, this can be related to the gas phase conformations [63]. Therefore, measurements of cross sections allow detection of gas phase protein folding and unfolding [46, 64, 100]. Energy loss experiments were used to determine these cross sections. The loss of kinetic energy from collisions of ions injected into the collision cell is measured. This loss of energy is then related to the collision cross sections through an aerodynamic drag model [101, 102]. Collisions of an ion with a gas molecule or atom can be modelled as specular (hard sphere elastic collisions) or diffuse (inelastic collisions). After a specular collision, the velocity components of the ion and the gas molecule parallel to the surface of the ion do not change, while the velocity component perpendicular to the surface is reversed. With diffuse scattering, the ion and a gas molecule scatter in an arbitrary direction with a cosine distribution around the normal to the surface of the ion, without any relation to the incident velocity direction [103]. The force F on an ion moving through a gas is related to the projection area A through a drag coefficient CD [102]: 32 2 dv Anm2v F=m,—=-CD (2.4) dt 2 where n is the gas number density, m, is the ion mass, m 2 is the collision gas mass, and v is the ion speed. The drag coefficient depends on: i) the density and viscosity of the gas, and ion speed (Reynolds number), ii) the ratio of the mean free path of a gas molecule to the ion size (Knudsen number), and iii) the ratio of the ion speed to the thermal speed of the gas. Drag coefficients have been calculated, for both specular and diffuse collisions, from the momentum transfer of a gas striking and leaving the surface of a sphere, integrated over all scattering angles and speeds [103]. If an ion enters a collision cell of length / with speed v0 and energy E°, and exits the cell with speed v and energy E, then integration and squaring of equation (2.4) gives the expression for the ion energy at the exit of the collision cell: E E° 2 f ^ ..... _,\ \voj exp\ CDnm2al m} j (2.5) where o is the collision cross section. Ions are collisionally cooled to kinetic energy spreads of 1 eV or less at the exit of Q0 [101, 104]. For collision cross section experiments, Q l was operated as an ion guide in RF only mode. Kinetic energies of ions leaving Q2 after collisions with argon were determined by increasing the rod offset voltage of Q3 until the ion signal was decreased 33 by several orders of magnitude, to generate stopping curves. The pressure of the collision gas was varied and stopping curves were obtained for each pressure. Energies E were taken as the energy at which the ion intensity was decreased to one tenth of the initial intensity, for each pressure [63]. The collision cross section for each charge state was E C YlYYl I determined from equation (2.5) by plotting - In—- versus — —. Drag coefficients E m, for diffuse scattering were used [101]. Typically drag coefficients were from 2.400 to 2.648. If specular scattering was used, these drag coefficients would be from 2.120 to 2.232. If specular scattering was used instead of diffuse scattering, collision cross sections would be 13-29% higher. This model was previously used to calculate collision cross sections of the following proteins and protein complexes: positive apomyoglobin, holomyoglobin and cytochrome c ions [101, 105], positive and negative ferro and ferrimyoglobin ions [106], lysozyme [107], bovine pancreatic trypsin inhibitor [108], and cytochrome c-cytochrome bs complexes [62]. In addition, the drag model has been used to calculate the collision cross sections of peptides: bombesin, bradykinin and luteinizing hormone releasing hormone [109]. 2.6 Tandem Mass Spectrometry Tandem mass spectrometry interpreted with a collision model was used to determine the additional internal energy, AEint, that must be added to the initial energy of the ion to 34 cause dissociation of the complex [62, 105]. This model, developed previously [62, 105], assumes that ions undergo many inelastic collisions in the collision cell. The collision model takes into account different numbers of collisions and the different kinetic energy losses of ions with different collision cross sections, as they move through the cell. In addition, at higher pressures, ions have more collisions with the gas and require lower injection voltages to cause dissociation. An ion of a complex at a distance d from the cell entrance has a lab kinetic energy given by: where m, is the mass of the ion of the complex, and all other variables are as above. The mean free path of the ion is given by: and therefore after traveling a distance Ad , the number of collisions the ion experiences is: (2.6) (2.7) N = Ad = no~ Ad (2.8) X 35 The maximum internal energy an ion can gain in the collision cell is the center of mass (CM) energy of each collision, summed over all collisions. If 0 is the average fraction of the C M kinetic energy transferred to internal energy in a single collision (taken as 0 = 1.0) [100], then the increase in the internal energy in traveling the distance Ad is: CDnm2ad AEint=0ECMN = 0^EnaAd=0^E°e m' na Ad (2.9) M M where ECM is the C M kinetic energy of an ion in a collision, and M = m, +m 2 . If equation (2.9) is written in a differential form and integrated over the cell length / , then the total internal energy added from all the collisions, can be written as: Cnnm7al AE;. 0-ffl, m. M m2 CD 1-e (2.10) The ion of interest was mass selected in Q l and injected into the collision cell, where multiple collisions with argon caused dissociation. Fragment ions were mass analyzed in Q3. The Q0-Q2 rod offset difference, which determines the kinetic energy of an ion at the cell entrance, was systematically increased in order to determine the voltage difference required to cause complexes to dissociate. The Q3 rod offset was set equal to the Q2 rod offset. For most complexes, where there is only loss of a neutral inhibitor, the dissociation voltage is taken as the Q0-Q2 voltage difference giving a 50% (arbitrary value) loss of the inhibitor from the complex. 36 Internal energies needed for dissociation were calculated for all complexes at different pressures of the collision gas, ranging from 0.5 mtorr to 2.74 mtorr. At different collision cell pressures, ions have different dissociation voltages and their speeds differ. Therefore, at different pressures, ions have different reaction times. It was previously found by simulations that under the conditions that give 50% reaction yield at the cell exit, reaction occurs over a length / ' , of about 5 cm near the cell exit, about 18 cm from the entrance [62]. The ion speed in this region can be calculated from equation (2.5), using the translational energy near the cell exit with /= 18 cm. This gives the expression for the reaction time as follows: r, = (2.11) (2E° -expl CDnm2la m where /' = 5 cm. The times available for reaction to occur were calculated for all complexes, since the internal energies needed to induce dissociation, are best compared for equal reaction times. A reaction time of 25 us was used to compare added internal energies, A E i n t , that cause 50% dissociation. 37 2.7 Hydrogen/Deuterium Exchange Labile hydrogens are found on carboxyl (COOH), hydroxyl (OH), amino (NH2) and thiol (SH) groups, and non-labile hydrogens are those bonded to carbon [110]. The labile hydrogens are exchangeable by deuterium, while non-labile hydrogens cannot be exchanged [111]. Hydrogens on the amino acid side chains, as well as hydrogens from the backbone amide positions exchange. The total number of all exchangeable hydrogens on the protein is the maximum H/D exchange level. For a protein in solution, the number of hydrogen atoms that can exchange with deuterium, or the H/D exchange level, is an indicator of protein conformation and dynamics, since the exchange will occur only when hydrogens are exposed to the solvent [112, 113]. It has been found that folded conformations exchange fewer hydrogen atoms than unfolded conformations, because in folded conformations, some exchangeable hydrogens are buried inside the protein [112, 114-118]. Mass spectrometry is a suitable method for determining the number and rates of exchange with deuterium from the solvent, since each incorporated deuterium results in a 1 Da increase in the total mass of a protein. If there are two protein conformations in solution that exchange at different rates, there are two possible mechanisms of H/D exchange [117]. In the EX1 mechanism, the two protein conformations interconvert more slowly than the exchange rate and, after exchange, two peaks per charge state appear in the mass 38 spectrum. In the EX2 mechanism, the two conformations interconvert more rapidly than the exchange rate and only one peak per charge state is observed in the mass spectrum. Hydrogen/deuterium exchange experiments were done under the same conditions as the MS experiments described in section 2.1. All experiments were done at room temperature, 23 °C. The solutions of enzyme and complexes were prepared as described in section 2.3. Instead of diluting the proteins and their complexes in deionized water and methanol, D 2 0 and C D 3 O D were used. The solutions were rapidly loaded into the syringe and injected into the mass spectrometer at a flow rate of 1 pL/min. Mass spectra of the + 10 and +11 charge states were simultaneously recorded for each solution at different exchange times. The H/D exchange level was calculated by subtracting the m/z of the peak at the beginning of the experiment (time = 0 min) from the m/z of the peak at a particular exchange time as exchange progresses, and multiplying the result by the charge. The H/D exchange level was plotted against the exchange time. 39 Chapter 3 Mass Spectra of Cex and its Complexes 3.1 Spectra of Cex In their native state, globular proteins are tightly folded, forming compact structures. When subjected to extreme temperatures or p H values, detergents, or solutions containing high concentrations of organic solvents, they become denatured, and therefore unfolded [31, 119-121]. These two different protein conformations can be detected by E S I - M S in positive ion mode, since the mass spectra of positive ions are strongly dependent on the folding state of the protein. ESI charge state distributions can be used to detect changes in the tertiary structure of proteins. Different protein conformations can generate mass spectra with different charge state distributions. There are two possible reasons for this. Unfolded proteins have more sites available for protonation and therefore exhibit higher charge states and broader charge state distributions [121, 122]. In addition, an unfolded form of a protein has a larger surface area than the folded form [10]. Native-like or folded proteins typically exhibit lower charge states and narrower charge state distributions of positive ions than the denatured proteins. Negative ion mass spectrometric studies of protein folding and unfolding led to two different observations. In some cases, like calbindin D - 2 8 K [37], calmodulin [123], or DNA-binding domain of the vitamin D 40 receptor [124], the unfolding of the protein causes broad charge state distributions with high charge states, similar to the charge state distributions obtained in positive ion mode. In other cases, like ubiquitin or cytochrome c [119], unfolding of proteins has a minor or no effect on negative ion charge state distributions. 600 800 1000 1200 1400 1600 1800 2000 m/z Figure 3.1 Mass spectrum of a solution of denatured WT Cex in 10% methanol and 1% acetic acid (pH 2.3). Figure 3.1 shows a mass spectrum of 10 pM WT Cex in 10% methanol and 1% acetic acid, at pH 2.3. At this pH, WT Cex is denatured and shows a broad charge state distribution, with high charge states from +20 to +42, with +35 being the most intense peak. A series of spectra of denatured Cex, like the one in Figure 3.1, were used for determination of the molecular mass of Cex, described in section 3.3. 41 The optimum pH range for the catalytic activity of xylanases is from 4 to 7 [79]. In this range, Cex is in its native conformation. Figure 3.2 shows a mass spectrum of 10 uM WT Cex in 10% methanol and 10 mM ammonium acetate buffer, at pH 5.8. The distribution of charge states is narrower, ranging from +9 to +13, with +11 being the most intense peak. The mass spectrum shows that Cex is in a more folded state. This is expected, since Cex is in its native state at this pH. 100 c > a DC Figure 3.2 Mass spectrum of a solution of native WT Cex in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8). 42 3.2 Spectra of Complexes Figure 3.3 shows a mass spectrum of a solution of WT Cex and X 2 DNJ in 10% methanol and 10 mM ammonium acetate buffer, at pH 5.8. The calculated concentrations of the enzyme, inhibitor and complex are 1, 29, and 4 pM, respectively. Peaks from enzyme (Enz ) and complex (Enz • Inh) are seen. A narrow distribution of charge states from +9 to +12, with +11 being the most intense, is formed, as expected for the protein in its native conformation. The peaks of the complex are 30-40% as intense as those of the 100 „ 80 -\ CD > CD CC 60 40 H 20 H + 1 lEnz + 12Enz-lnh + 12Enz/1 +HEnz-Inh + 10Enz 2800 3000 + 10Enz-Inh +9 Enz-Inh +10Enz-lnh2 +9Enz ^ 3600 3800 4000 Figure 3.3 Mass spectrum of a solution of WT Cex and X 2 DNJ in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8); [Enz] = 1 pM, [inh] = 29 pM, [Enz • Inh] = 4 pM. 43 enzyme for the same charge state, even though, from the solution/^., it is calculated that almost all the enzyme was in the complexed form. Therefore, the gas phase abundances do not reflect the solution equilibria. The lower abundance of ions of the complex may be the result of the addition of 10% methanol to the solution, some dissociation of the complex in the orifice-skimmer region, or a shift to lower pH during the spray process. A third low intensity peak for each charge state appears in the spectrum, corresponding to the mass of Enz • Inh2 . Since neither WT Cex nor its mutants have more than one binding site, this peak is attributed to non-specific binding. 100 80 -i—i _c CD > j | 40 CD DC 20 + 12Enz "^ii 11 11 + 11 Enz + 11'Enz-lnh + 10Enz +10Enz-lnh + 10Enz-Inh2 +9Enz-Inh +9Enz f1 2800 3000 3200 3400 3600 3800 4000 m/z Figure 3.4 Mass spectrum of a solution of WT Cex and X 2 IL in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8); [Enz] = 0.04 uM, [inh] = 43 uM, [Enz • Inh] = 4.9 uM. 44 With all the forms of Cex at pH 5.8, spectra showed narrow charge state distributions with charges +9 to +12. Spectra of the complexes with X2IL always had +11 as the most intense peak, as with complexes with X 2 D N J . A spectrum of WT Cex with X 2 fL in 10% methanol and 10 nM ammonium acetate buffer, at pH 5.8, is shown in the Figure 3.4. The calculated concentrations of the enzyme, inhibitor and complex were 0.04, 43, and 4.9 pM, respectively. 100 A 80 CO g 6 0 CD > CD DC 40 20 + 10Enz + ]2Enz A + 11 Enz + ] lEnz-lnh + 10Enz-Inh 2800 3000 3200 3400 m/z 3600 +9Enz-Inh +9Enf\jt 3800 4000 Figure 3.5 Mass spectrum of a solution of WT Cex and X 2IF in 10% methanol and 10 mM ammonium acetate buffer (pH 5.8); [Enz] = 0.02 pM, [inh] = 34 pM, [Enz • Inh] = 4.9 pM. A spectrum of the complex of WT Cex with X 2IF in 10% methanol and 10 pM ammonium acetate buffer, at pH 5.8, is shown in the Figure 3.5. The calculated 45 concentrations of the enzyme, inhibitor and complex were 0.02, 34, and 4.9 uM, respectively. In the spectra with X2IF, the most intense peak is +10. Spectra of the mutants were slightly shifted in mass compared to WT Cex, corresponding to the difference in mass of the native and altered amino acid residues. The charge states of the complexes with mutants were the same as of WT Cex for a given inhibitor, suggesting that the charge state distribution depends somewhat on the inhibitor. 3.3 Mass of Cex Both wide and narrow charge state distributions of a protein in mass spectra allow the determination of the molecular mass of the protein. If a series of peaks in a mass spectrum represents different protonation states, then the m/z for two neighbouring peaks, x, and x2, are defined by following expressions: x, = Mp+z M p + z + l ' z + 1 (3.1) (3.2) where Mp is the molecular mass of the protein, and z is the charge state of an analyte ion. Based on equations (3.1) and (3.2), the molecular mass can be calculated using the m/z ratios of any two adjacent peaks: 46 X j X2 In practice, the calculation of mass is performed by a computer, including all peaks in the spectrum. The production of the catalytic domain of Cex involves the in vivo post-translational removal of the Cex N-terminal leader peptide, as well as the in vitro cleavage and removal of a C-terminal linker and cellulose-binding region. In comparison to the published sequence from which the crystal structure was derived [76], the catalytic domain of Cex, produced and used in experiments presented in this thesis, contains the additional amino acids GlyAlaSer at the C-terminus, and contains the residues ValValLysProAlaGlnAla at the N-terminus to give a calculated molecular mass of 34,849 Da. Based on the mass spectrum of denatured Cex, shown in Figure 3.1, reproduced six times, a mass of 34,866 ± 44 Da was determined. This experimentally determined mass is in reasonable agreement with the calculated mass. 47 Chapter 4 Collision Cross Sections 4.1 Stopping Curves In collision cross section experiments, the stopping potential is the potential difference between the Q3 and Q2 rod offsets (Q2 was kept at 100 V in all collision cross section experiments). As the Q3 potential is increased, ions show a loss of intensity. The energy to stop ions was taken as the stopping potential at which the ion intensity was reduced to one tenth of the initial intensity, multiplied by the charge of the ion. Stopping curves, obtained as described in Section 2.4, are shown in Figure 4.1 for the +10 charge state of WT Cex. Stopping curves for six different pressures of argon in the collision cell are shown: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mtorr. They are sharp, with one order of magnitude decrease in ion intensity with 1-2 V increase in stopping potential. The initial energy E° (the energy at the cell entrance) was taken as the energy at one tenth of the intensity when there is no collision gas in the cell, while E (the energy at the cell exit) was taken as the energy at one tenth of the intensity for each pressure of the collision gas. Drag coefficients were determined from the plot CD - f(s) for diffuse 48 scattering, where s is the ratio of the speed of the ion to the thermal speed of the gas [103]. 0 mtorr — o — 0.3 mtorr —•— 0.6 mtorr v — 0.9 mtorr —•— 1.2 mtorr 1.5 mtorr 1e + 5 A c o> o 1 e + 3 H i i i i— 100 105 110 115 120 Q3 P o t e n t i a l ( V ) Figure 4.1 Stopping curves for the +10 charge state of WT Cex at different pressures of argon. For each set of stopping curves, a graph - In—- vs. can be plotted. This type of E m, plot for the +10 charge state of WT Cex is shown in Figure 4.2. From equation (2.5), it is evident that the collision cross section a can be determined from the slope of this linear plot. In Figure 4.2 the slope is 1.778, giving a cross section of 1778 A 2 . For this plot, R2 value is 0.9992, indicating a good fit to the data points. The intersection on y axis is - 0. 0009. 49 0.5 Figure 4.2 Linear plot of - In — vs. —-—— for the +10 charge state of WT Cex. The E m, collision cross section a is determined by the slope of the line (the slope is 1.7783 according to the fit). 4.2 Collision Cross Sections of WT Cex and Its Complexes Collision cross sections were measured for Enz and Enz • Inh ions from solutions of all the complexes, and for the Enz ions from solutions in which only protein was present. Table 4.1 lists all the cross sections obtained for WT Cex and its complexes. Uncertainties are standard deviations of four replicate measurements. 50 WT WT-X 2DNJ WT-X 2IL WT-X 2IF charge state Enz Enz Enz-Inh Enz Enz-Inh Enz Enz-Inh +9 1727+31 1320+91 1398±44 1348+69 1428±36 1546±37 1218+44 +10 1790±49 1362+61 1378+15 1444±10 1448±29 1636±55 1257±26 +11 1803±44 1591±68 1552+38 1578+73 1526±36 1637+33 1318+65 Table 4.1 Collision cross sections (A2) of WT Cex and WT complexes. The labels in the first row indicate the solution used, either protein alone, or protein and inhibitor. There is a small increase in the cross sections with the charge state of the ions, although in some cases this increase is less than the combined uncertainties in the cross sections. This is likely the result of Coulomb repulsion within the ions, causing them to unfold. Free protein ions formed from solutions in which only enzyme is present have slightly, but consistently, higher cross sections for a given charge state than free protein ions from solutions of the complexes of the same protein. This difference is significant for the WT Cex, which can be seen in Figure 4.3. It is possible that in the presence of the solution complex, some protein ions are formed by dissociation of the complex in the orifice-skimmer region. If protein ions retain the same conformation as in the complex on the time scale of the experiment, they may retain more compact configurations. Ions of the complexes may be dissociated in the orifice-skimmer region by energetic collisions that "heat" the complexes. Although ions are cooled when passing through the skimmer to form a supersonic jet, increase in the orifice-skimmer voltage difference increases the energy given to the ions in this region and the overall effect is heating of the ions. If 51 energy that would otherwise cause the protein ions to unfold somewhat is used to dissociate the complexes, less energy is available to cause the protein ions to unfold, leaving them with slightly more compact structures. • Enz from W T - X 2 I L O — Enzlnh from W T - X 2 I L A Enz from W T - X 2 I F A. — Enzlnh from W T - X 2 I F T 1 1 9 10 11 C h a r g e S t a t e Figure 4.3 Collision cross sections vs. charge state for ions from a solution of WT Cex only and Enz and Enz • Inh ions from the solutions of the complexes of WT Cex with X 2 DNJ,X 2 IL and X 2IF. Ions of the complexes consistently have slightly lower cross sections than protein ions of the same charge state formed from solutions of protein only, suggesting that binding the inhibitors helps to stabilize the protein in more compact structures. This is discussed further in Chapter 5.5. 52 5000 1000 A 5 10 15 20 25 30 35 40 Charge State Figure 4.4 Collision cross sections vs. charge state for native and denatured WT Cex. Collision cross sections of ions of denatured WT Cex were also measured. Figure 4.4 is a plot of collision cross section versus charge state for all charge states. Ions formed from denatured WT Cex (charges +20 to +42) have more open conformations than ions from native WT Cex (charges +9 to +12). Proteins lose their compact ordered structures upon denaturation and their more open conformations have more sites available for protonation. The large number of charges on the denatured proteins causes stronger Coulomb repulsion which causes unfolding of the gas phase ions. The cross sections of ions from denatured WT Cex are about two times greater than the cross sections of ions from native WT Cex. From the cross sections of ions from denatured WT Cex, the increase of collision cross section with charge state is more evident. 53 4.3 Collision Cross Sections of Mutants and Their Complexes Collision cross sections of mutants and their complexes are listed in Tables 4.2-4.4. Plots of collision cross sections of mutant proteins and their complexes vs. charge state are shown in Figures 4.5-4.7. As with WT Cex, for all Enz and Enz • Inh ions, the cross sections increase somewhat with the charge state of the ion. Ions from solutions that contain only mutant protein have slightly lower cross sections than ions of the same charge state of WT Cex (within the uncertainties, except for the +9 charge state), suggesting somewhat more compact conformations. This is illustrated in Figure 4.8 (note that the y axis scaling is different than in the previous plots). However, the cross sections of complexes with mutants are somewhat higher than the cross sections of complexes of WT Cex with the corresponding inhibitors. This suggests that the binding of the inhibitor stabilizes WT Cex complexes to a greater extent than mutant complexes. This is discussed in more detail in Chapter 5.5. Gln87Met Gln87Met -X 2DNJ Gln87Met -X 2EL Gln87Met -X 2IF charge state Enz Enz Enz-Inh Enz Enz-Inh Enz Enz-Inh +9 1620±64 1529±34 1412+53 1548+32 1403±21 1432±56 1262±22 + 10 1720±72 1548±56 1486±48 1590+36 1526±46 1476±28 1354±46 + 11 1780±46 1620±23 1595+81 1635+74 1582+36 1498±43 1426±33 Table 4.2 Collision cross sections (A2) of Gln87Met and Gln87Met complexes. 54 Gln87Tyr Gln87Tyr -X 2DNJ Gln87Tyr -X 2 IL Gln87Tyr -X 2IF charge state Enz Enz Enz-lnh Enz Enz-lnh Enz Enzlnh +9 1642±46 1573±32 1562+36 1554+33 1554±87 1465±85 1437±48 +10 1670±42 1638+57 1595±51 1654±100 1601±52 1450±72 1408±58 + 11 1762±50 1630±43 1606+51 1722±83 1599±44 1518+44 1475±49 Table 4.3 Collision cross sections (A2) of Gln87Tyr and Gln87Tyr complexes. Asn44Ala Asn44Ala -X 2DNJ Asn44Ala -X 2 IL Asn44Ala -X 2IF charge state Enz Enz Enz-lnh Enz Enz-lnh Enz Enz-lnh +9 1605±44 1591+32 1581+22 1598+33 1518+18 1522±72 1422±44 +10 1638+31 1636+98 1612+69 1655+50 1618+58 1562±27 1486±70 +11 1730+12 1701+33 1685±74 1699±39 1605±46 1582±43 1482±62 Table 4.4 Collision cross sections (A2) of Asn44Ala and Asn44Ala complexes. 55 1800 1600 g o cu c/> CO CO 2 1400 H c g ]co o O 1200 H 1000 GIn87Met only Enz f rom G l n 8 7 M e t - X 2 D N J Enz f rom Gln87Met-X 2 IL Enzlnh from Gln87Met-X 2 IL Enz f rom Gln87Met -X 2 IF Enzlnh from Gln87Met-X, IF — i — 10 11 C h a r g e S t a t e Figure 4.5 Collision cross sections vs. charge state for ions from the solutions of Gln87Met only and Enz and Enz • Inh ions from the solutions of its complexes. 1800 H § 1600 o CD CO CO 2 1400 H c o c^o "o O 1200 1000 Gln87Tyr only Enz f rom G l n 8 7 T y r - X 2 D N J Enzlnh from G l n 8 7 T y r - X 2 D N J Enz f rom Gln87Tyr -X 2 IL Enzlnh from Gln87Tyr -X 2 IL Enz f rom Gln87Tyr -X 2 IF Enzlnh from Gln87Tyr -X 2 IF 10 11 C h a r g e S t a t e Figure 4.6 Collision cross sections vs. charge state for ions from the solutions of Gln87Tyr only and Enz and Enz • Inh ions from the solutions of its complexes. 56 q +-» o CD co CO CO o 6 c g 'co ~o O 1 8 0 0 1 6 0 0 H 1 4 0 0 1 2 0 0 1 0 0 0 A s n 4 4 A l a o n l y E n z f r o m A s n 4 ^ Enzlnh from A s n 4 4 A l a - X 2 D N J Enz f r om A s n 4 4 A l a - X 2 I L Enzlnh f r o m A s n 4 4 A l a - X 2 I L E n z f r om A s n 4 4 A l a - X 2 I F 10 11 C h a r g e S t a t e Figure 4.7 Collision cross sections vs. charge state for ions from the solution of Asn44Ala only and Enz and Enz • Inh ions from the solutions of its complexes. c o o CD CO CO co O O c o ]co o O 1 9 0 0 1 8 0 0 1 7 0 0 H 1 6 0 0 H 1 5 0 0 1 4 0 0 WT Cex only Gln87Met only Gln87Tyr only Asn44Ala only 10 C h a r g e S t a t e — i — 11 Figure 4.8 Collision cross sections vs. charge state for ions from the solutions of proteins only. 57 For all the proteins, WT Cex and the mutants, the complexes with X2EF always have the lowest cross sections, while complexes with X 2 D N J always have the highest cross sections. The binding strengths in solution increase in the order X 2 DNJ < X 2 IL < X 2IF. This correlation between the gas phase cross sections and binding strengths in the solution will be discussed further in Chapter 5. 58 Chapter 5 Binding Studies of Gas Phase Ions 5.1 MS/MS Spectra Most complexes were found to dissociate simply via loss of a neutral inhibitor. As the collision energy is increased the intensity of the (Enz • Inh)+n peak in the MS/MS spectrum decreases, accompanied by an increase in the intensity of the Enz+n peak. Relative intensities of the precursor (complex) arid fragment (enzyme) ions were calculated from: (%) = 1 precursor m % ( 5 ] ) precursor \ ' J j j ,~ v - , precursor fragment W > ) = Ifm8mT 100% (5.2) ^precursor ^fragment where I rsor is the intensity of the precursor, Ifragment is the intensity of the fragment (complex minus neutral inhibitor). Figure 5.1 shows MS/MS spectra for the dissociation of the +10 charge state of the complex of WT Cex with X 2IF at a collision cell pressure 59 E = 1 5 0 e V + 10 Enz-lnh J \ + 10 Enz E = 1 5 5 0 e V +10 Enz-lnh E = 9 5 0 e V + 10 Enz + 10 Enz-lnh + 10 Enz E = 1 6 5 0 e V + 10 Enz-lnh E = 1 5 2 0 e V + 10 Enz + 10 Enz-lnh E = 2 1 5 0 e V -10 Enz + 10 Enz-lnh 3800 3200 Figure 5.1 MS/MS spectra of +10 WT Cex with X 2IF ions at a collision cell pressure of 1.5 mtorr, at ion initial kinetic energies of 150, 950, 1520, 1550, 1650 and 2150 eV. 60 + 77 Enz + 11 Enz-lnh o -100 -„ 80 A E = 165 eV + 10 Enz + 11 Enz +11 Enz-lnh E = 1265 eV + 10 Enz + 77 Enz o • 100 -+ 77 Enz-lnh E = 605 eV + 10 Enz + 11 Enz +11 Enz-lnh E = 1375eV + 10 Enz _J4 I m 40 - + 7 7 Enz + 11 Enz-lnh E = 935 eV + 10 Enz + 7 7 Enz +11 Enz-lnh E = 1485 eV + 10 Enz 3300 3400 m/z 3500 3100 3300 3400 m/z Figure 5.2 MS/MS spectra of +11 WT Cex with X 2 DNJ ions at a collision cell pressure of 0.8 mtorr, at ion initial kinetic energies of 165, 605, 935, 1265, 1375 and 1485 eV. 61 of 1.5 mtorr, while Figure 5.2 shows MS/MS spectra for the dissociation of the +11 charge state of the complex of WT Cex with X 2 DNJ at a collision cell pressure of 0.8 mtorr. From Figure 5.1 it can be seen that the complex of WT Cex with X 2IF shows mostly neutral inhibitor loss, with less than 2% of charged inhibitor loss. Neutral inhibitor loss for the+10 charge state of the complex can be represented by: Enz • Inh+1° -» Enz+W + Inh0 (5.3) All the complexes of mutant proteins with all three inhibitors showed only neutral inhibitor loss. Two complexes show different behaviour, WT Cex with X 2 DNJ and WT Cex with X 2 IL. Figure 5.2 shows that the complex of WT Cex with X 2 DNJ shows both neutral and charged inhibitor loss, which for the +11 charge state of the complex can be represented by: Enz • Inh+n -* Enz+n + Inh0 (5.4) Enz • Inh+" -> Enz+W + Inh+1 (5.5) The amounts of charged inhibitor loss for complexes of WT Cex with both X 2 DNJ and X 2 IL are small, less than 20%. The ratio of charged-to-neutral inhibitor loss was largely independent of collision energy, indicating that similar energies are required for dissociation by either channel. 62 5.2 Dissociation Curves Dissociation curves are plots of relative intensities of precursor and fragment ions versus Q0-Q2 voltage differences. Figure 5.3 shows the dissociation curve for the +10 charge state of the complex of WT Cex with X2IF at a cell pressure of 1.5 mtorr. The dissociation voltage is taken as the voltage at 50% precursor loss or the point where the two curves intercept. +10 Enz-Inh Q0-Q2 (V) Figure 5.3 Relative abundances of the +10 Enz-Inh and +10 Enz ions in MS/MS spectra of WT Cex-X2lF ions vs. Q0-Q2 rod offset voltage difference at a collision cell pressure of 1.5 mtorr of Ar. 63 The dissociation curve for the +11 charge state of the complex of WT Cex with X 2 DNJ at a collision cell pressure of 0.8 mtorr is shown in Figure 5.4. 200 Q0-Q2 (V) Figure 5.4 Relative abundances of the +11 Enz-lnh, +11 Enz and +10 Enz ions in MS/MS spectra of WT Cex-X2DNJ ions vs. Q0-Q2 rod offset voltage difference at a collision cell pressure of 0.8 mtorr of Ar. The data for this complex were analyzed in two ways. First, only the neutral loss channel was included, and the dissociation voltage was determined as for the complex of WT Cex with X 2IF. Second, the sum of charged and neutral losses was plotted versus energy, and the voltage where the intensity of + z Enz • Inh ion equalled the sum of the intensities of + zEnz and +(z-1 )Enz ions was taken as the dissociation voltage. This second 64 method produced dissociation voltages that were 2-11% lower than the first. The dissociation voltages calculated from the second method were used in this thesis. Figure 5.5 shows the dissociation curve constructed using the second method for the same data shown in the Figure 5.4. 0 5 0 1 0 0 1 5 0 2 0 0 Q0-Q2 (V) Figure 5.5 Relative abundances of the +11 Enz-Inh, and the sum of +11 Enz and +10 Enz ions in MS/MS spectra of WT Cex-X2DNJ ions vs. Q0-Q2 rod offset voltage difference at a collision cell pressure of 0.8 mtorr of Ar. The dissociation voltages for the complex of WT Cex with X 2 IL, which also showed both neutral and charged inhibitor losses, were calculated in the same way as described for the complex of WT Cex with X 2 DNJ. 65 5.3 Dissociation at Different Collision Cell Pressures Dissociation voltages were determined for the +10 and +11 charge states of all the complexes at collision cell pressures ranging from 0.50 to 2.74 mtorr. At lower pressures, higher voltages are required for dissociation because ions have fewer collisions and therefore more energy per collision is required. Energy losses of ions moving through the cell must be considered and losses are lower at lower pressures. In addition, at lower pressures, ions travel faster through the cell, have less time available for reaction and therefore need more internal energy to give a 50% fragmentation yield, than at higher pressures. Dissociation voltages for +10 and +11 charge states of the complexes of WT Cex and the mutants at different collision cell pressures are shown in the Figures 5.6-5.9. In most cases +10 charge states for all complexes require higher dissociation voltages than +11 charge states. For a given pressure, complexes with X2IF require the highest, while complexes with X 2 D N J require the lowest dissociation voltages for all proteins. This has the same trend as the binding in solution, where the complexes with X2IF have the strongest, while complexes with X 2 DNJ have the weakest binding. 66 Figure 5.6 The dissociation voltages of the +10 and +11 charge states of the complexes of WT Cex with inhibitors X 2 DNJ, X 2 IL and X 2IF at different cell pressures. Complexes with mutant proteins require less voltage to dissociate than complexes with WT Cex for the same charge state and at the same collision cell pressure. This shows the same trend as the binding in solution, where binding of complexes with mutant proteins is weaker than binding of complexes of WT Cex for a given inhibitor. 67 3 0 0 o H 1 \ 1 1 1 1 0 .0 0 . 5 1.0 1.5 2 . 0 2 . 5 3 . 0 P r e s s u r e (mtorr ) Figure 5.7 The dissociation voltages of the +10 and +11 charge states of the complexes of Gln87Met at different cell pressures. 3 0 0 + 10 G l n 8 7 T y r - X 2 D N J ». — + 11 G l n 8 7 T y r - X 2 D N J © + 10 Gln87Tyr -X 2 IL &• — +11 Gln87Tyr -X 2 IL A + 10 G ln87Tyr -X 2 IF A - +11 Gln87Tyr -X 2 IF o > 0 H 1 1 1 1 1 1 0 .0 0 . 5 1.0 1.5 2 . 0 2 . 5 3 . 0 P r e s s u r e (mtorr ) Figure 5.8 The dissociation voltages of the +10 and +11 charge states of the complexes of Gln87Tyr at different cell pressures. 68 3 0 0 -| + 10 A s n 4 4 A l a - X 2 D N J + 11 A s n 4 4 A l a - X 2 D N J + 10 Asn44A la -X 2 I L + 11 Asn44A la -X 2 I L + 10 A s n 4 4 A l a - X 2 I F + 11 A s n 4 4 A i a - X 2 I F o > c 150 -0.0 0.5 1.0 1.5 2.0 2 .5 3.0 P r e s s u r e (mtor r ) Figure 5.9 The dissociation voltages of the +10 and +11 charge states of the complexes of Asn44Ala at different cell pressures. 5.4 Internal Energies and Reaction Times Internal energies needed to be added to the ions to cause the dissociation at each pressure were calculated using equation (2.10). Reaction times for each pressure were calculated using equation (2.11). For calculations of both added internal energy and reaction times, experimentally determined dissociation voltages and collision cross sections were used. Figure 5.10 shows a plot of A£ i n t versus reaction time for complexes of WT Cex. It can be seen that within the uncertainties, similar energies are required to dissociate the +10 and +11 charge states for each complex. The values of AEim for a given reaction time follow the order WT-X 2IF > WT-X 2IL > WT-X 2 DNJ. 2 5 0 0) 2 0 0 i a) 69 Figure 5.10 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of WT Cex. The curves of AEint vs. reaction time for the complexes with mutants are shown in Figures 5.11-5.13. They are similar to the curves for the complexes with WT Cex, although AE-M values are generally lower. The greatest internal energy is needed for the dissociation of complexes with X 2IF and the lowest for complexes with X 2 DNJ. The internal energies needed for the dissociation of all complexes with mutants are lower than for the corresponding complexes for WT. Among the mutants, Gln87Met requires the highest, Gln87Tyr less, and Asn44Ala the lowest energy for dissociation. This shows the same trend as the solution binding, where Gln87Met shows the strongest, Gln87Tyr weaker and Asn44Ala the weakest binding strengths with a given inhibitor. 70 Values of AZsint used below were taken from the plots in Figures 5.10-5.13 for equal reaction times of 25 ps (shown by a dashed line), since the best comparison of AEint values is for equal reaction times. Values of AEiat for dissociation of +10 and +11 ions of all the complexes in 25 ps are shown in Table 5.1. Also shown are values of the free energy change for dissociation of the complexes in solution at 37 °C, AG dissociation j calculated from AG dissociation -kBT\nKt, where kB is Boltzmann's constant (kB = 8.617 xlO'5 eV K~'), and T is temperature. 200 150 > CD * 100 i m 50 1e-5 2e-5 3e-5 4e-5 Reaction Time (s) + 10 G l n 8 7 M e t - X 2 D N J + 11 G l n 8 7 M e t - X 2 D N J + 10 G l n 8 7 M e t - X 2 I L + 11 G l n 8 7 M e t - X 2 I L + 10 G l n 8 7 M e t - X 2 I F + 1 1 G l n 8 7 M e t - X 2 I F 5e-5 6e-5 Figure 5.11 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of Gln87Met. 71 > CD uj •"3 2 0 0 1 5 0 H 100 H 5 0 + 10 G l n 8 7 T y r - X 2 D N J + 11 G l n 8 7 T y r - X 2 D N J + 10 G ln87Tyr -X 2 IL + 11 G ln87Tyr -X 2 IL + 10 G ln87Tyr -X 2 IF n87Tyr-X 2 IF 1 e - 5 2 e - 5 3 e - 5 4 e - 5 5 e - 5 R e a c t i o n T i m e (s) 6 e - 5 7 e - 5 8 e - 5 Figure 5.12 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of Gln87Tyr. > LU 2 0 0 1 5 0 1 0 0 5 0 + 11 A s n 4 4 A l a - X , D N J + 10 Asn44A la -X , IF 2 e - 5 4 e - 5 6 e - 5 R e a c t i o n T i m e (s) 8 e - 5 l e - 4 Figure 5.13 Added internal energies to cause dissociation, AEint vs. the time available for reaction for the +10 and +11 charge states of complexes of Asn44Ala. 72 COMPLEX dissociation (e^) +10 ions *Emt (eV) +11 ions *Eu« (eV) WT-X 2DNJ 0.318 86±3 95±3 WT-X 2IL 0.393 113±6 105±3 WT-X2IF 0.419 154±5 147±5 Gln87Met-X2DNJ 0.288 69±4 79±4 Gln87Met-X2IL 0.345 102±3 92±2 Gln87Met-X2IF 0.390 131±3 139±2 Gln87Tyr-X2DNJ 0.256 68±4 53±2 Gln87Tyr-X2IL 0.302 81±3 73±4 Gln87Tyr-X2IF 0.360 122±4 125±6 Asn44Ala-X2DNJ 0.228 45±3 48±3 Asn44Ala-X2IL 0.281 53±4 53±3 Asn44Ala-X2IF 0.313 95±4 93±4 Table 5.1 Free energy changes for the dissociation of complexes in solution and internal energies required for the dissociation of gas phase complexes in 25 ps. The complexes with two bound inhibitors were not studied in detail. A preliminary experiment with +11 WT-X 2 DNJ 2 ions showed that this complex dissociates by roughly equal losses of charged and neutral inhibitor. The internal energy required to reduce the precursor intensity to one half of its initial value was found to be AE i n t = 45 eV (0.8 mtorr cell pressure, 30 ps reaction time). This energy is about one half of that required for 73 dissociation of the +11 WT-X 2DNJ complex ions in about the same reaction time (95 eV, Table 5.1 and Figure 5.10), suggesting weaker binding of the nonspecific complex. This contrasts to the findings of Wang and coworkers where nonspecific binding was found to be stronger than specific binding in a protein-carbohydrate complex [125]. 5.5 Collision Cross Sections and Gas Phase Binding The cross sections and AE i n t values show that binding of inhibitors more strongly in the gas phase complex causes the protein to adopt more compact conformations. Figures 5.14 a) and 5.14 b) show cross sections for all the complexes versus the AEiBt values for dissociation of the complexes, for the +10 and +11 ions respectively. Also shown in Figure 5.14, by dashed lines, are the cross sections of the protein ions produced from solutions of protein only. All complexes have cross sections lower than those of the protein ions formed from solutions of protein only. The complexes and protein ions in these low charge states retain considerable folded structures; apo-protein ions with charge states +22 to +35 formed from Cex denatured in solution have considerably greater cross sections of 3300 to 4000 A 2 (Figure 4.4). There is a strong correlation between the cross sections and Ais in t values, with the more strongly bound complexes having smaller cross sections. The lines in Figure 5.14 are second order fits, simply to illustrate the trend. Binding the inhibitor apparently helps keep the protein folded, most likely around the binding pocket, with stronger binding giving more tightly folded conformations. A related effect was seen with gas phase holomyoglobin ions [126]. 74 2 0 0 0 1 8 0 0 -L g I 1 6 0 0 co CO o O o Ml o O 1 4 0 0 H 1 2 0 0 1 0 0 0 | N44A-X 2 IL ^ / T N 4 4 A - X o D N J / -L Q 8 7 M - X 2 D N J W T - X o D N J Q 8 7 Y - X 2 D N J N44A-X, IF Q87M-X 2 I F h W T - X 2 I F 4 0 6 0 — I — 8 0 WT Q87M Q87Y N44A 1 0 0 1 2 0 1 4 0 1 6 0 2 0 0 0 1 8 0 0 c: g 8 1 6 0 0 CO co co O o c o |w "o O 1 4 0 0 1 2 0 0 1 0 0 0 b) j N 4 4 A - X 2 D N J ^ Q 8 7 M - X 2 D N J Q87M-X 2 I L XoDNJ W T - X , I L N44A-X 2 IL Q 8 7 Y - X 2 D N J Q 8 7 M - X 2 I F W T - X 2 I F 4 0 — I — 6 0 — i — 80 W T Q87M Q87Y N44A 1 0 0 AEM (eV) 1 2 0 1 4 0 1 6 0 Figure 5.14 Collision cross sections of the complexes vs. the added internal energy to cause dissociation, AE=. , for a) +10 ions and b) +11 ions. 75 The change in average conformation with inhibitor binding is readily detected in the gas phase ions. In contrast, the crystal structures show no significant structural changes of the protein upon binding of the inhibitors [82-84]. However proteolysis and thermal denaturation studies show that the formation of a covalent glycosyl-enzyme intermediate stabilizes Cex against unfolding [127]. These observations suggest that, rather than a distinct different conformation being stabilized, the average conformation of the enzyme is reduced in size in the presence of the inhibitor. 5.6 Comparison of Solution and Gas Phase Binding 5.6.1. Solution H/D Exchange In order to further compare gas phase and solution conformations, solution H/D exchange experiments were performed. The total number of exchangeable hydrogens is calculated as the sum of amide hydrogens from the protein backbone and exchangeable hydrogens on the amino acid side chains. The total number of exchangeable hydrogens for all the proteins and complexes are presented in Table 5.2. Hydrogen/deuterium exchange level was determined at different exchange times. Typical curves of H/D exchange level vs. time, obtained for all complexes, are similar to the curve presented in the Figure 5.15 for the +10 and +11 charge states of the complex of WT Cex with X2IL. Hydrogen/deuterium exchange for the +10 and +11 charge states of 76 each of the complexes was recorded at the same time. It can be seen that the initial exchange rate for the +10 charge state of the complex is lower than the initial exchange rates of all other ions from the same solution. This was observed for most of the investigated ions. The saturation time was defined as the time where the maximum H/D exchange level was obtained, or after which there is almost no further exchange. The maximum H/D exchange level and the saturation times for all the ions are presented in Table 5.3. Protein or Complex Exchangeable hydrogens Protein or Complex Exchangeable hydrogens WT Cex 497 Gln87Tyr 496 WT-X 2DNJ 497 Gln87Tyr-X2DNJ 497 WT-X 2IL 496 Gln87Tyr-X2IL 497 WT-X2IF 496 Gln87Tyr-X2IE 495 Gln87Met 495 Asn44Ala 495 Gln87Met-X2DNJ 496 Asn44Ala-X2DNJ 498 Gln87Met-X2IL 495 Asn44Ala-X2IL 496 Gln87Met-X2IF 495 Asn44Ala-X2IF 496 . Table 5.2 Total numbers of exchangeable hydrogens for WT Cex, Gln87Met, Gln87Tyr and Asn44Ala and their complexes with X2DNJ, X 2 IL and X 2IF. 77 400 300 CD > CD _ l CD O) CO _c o X LU Q X 200 100 • o r ° 7cP o O o — I — 20 T 8 • V — I — 40 Time (min) o T . O • +10 Enz O +10 Enzlnh • +11 Enz V +11 Enzlnh — i — 60 Figure 5.15 H/D exchange levels vs. time of the +10 and +11 charge states of Enz and Enz • Inh ions of the complex of WT Cex with X2IL. From Table 5.3 it can be seen that Enz • Inh ions show consistently lower maximum H/D exchange levels than Enz ions from the same complex. This difference (about 40-50 hydrogens) is greater than the difference between calculated total possible numbers of exchangeable hydrogens for these ions from Table 5.2. This suggests that, in solution, the Enz • Inh complexes are more protected from the exchange with deuterium than free Enz , not only because the inhibitor protects some of the hydrogens upon binding, but because the protein takes somewhat more compact conformations upon inhibitor binding as well. More compact conformations upon binding were observed in the gas phase ions from collision cross section measurements, described in Chapter 4. Preliminary results a) + 10 ions +11 ions Protein or Complex Maximum H/D exchange level Saturation time (min) Maximum H/D exchange level Saturation time (min) WT Cex 238 ±6 6.4 ±0.3 217±6 7.6 ± 0.4 WT-X 2 DNJ Enz 327 ±6 9.9 ±0.9 305 ±8 8.7 ±0.2 Enz-lnh 270 ±7 11.5 ±0.7 288 ±7 9.7 ±0.8 WT-X 2IL Enz 303 ±9 12.2 ±0.8 339 ±6 11.6 ±0.3 Enz-lnh 259 ±8 13.2 ±0.9 315 ± 7 12.8 ±0.6 WT-X2IF Enz 316±4 12.3 ±0.6 334 ±4 13.8 ±0.6 Enz-lnh 267 ±3 17.9. ±0.8 301 ±7 15.3 ±0.3 b) +10 ions +11 ions Protein or Complex Maximum H/D exchange level Saturation time (min) Maximum H/D exchange level Saturation time (min) Gln87Met 245 ±6 10.0 ±0.4 231 ± 13 9.0 ±0.6 Gln87Met-X 2 DNJ Enz 330 ±8 9.7 ±0.7 307 ±3 9.0 ±0.5 Enz-lnh 265 ±5 11.6 ±0.6 288 ±5 9.6 ±0.5 Gln87Met-X 2 IL Enz 299 ±7 13.1 ±0.4 339 ± 1 12.1 ±0.3 Enz-lnh 256 ±2 16.0 ±0.4 278 ±5 14.7 ±0.2 Gln87Met-X 2IF Enz 308 ± 1 17.0 ±0.3 337 ±7 17.9 ±0.9 Enz-lnh 266 ±2 21.2 ±0.6 290 ± 2 20.3 ± 0.3 79 c) + 10 ions +11 ions Protein or Complex Maximum H / D exchange level Saturation time (min) Maximum H / D exchange level Saturation time (min) Gln87Tyr 219 ± 8 6.9 ± 0.4 202 ± 8 7.6 ±0.2 Gln87Tyr- Enz 341 ±2 9.2 ±0.6 310±2 9.3 ±0.3 X 2 D N J Enz-Inh 273 ±7 10.1 ±0.4 291 ± 2 10.9 ±0.6 Gln87Tyr- Enz 294 ±5 13.0 ±0.6 337 ± 2 13.1 ±0.7 X 2 I L Enz-Inh 272 ±9 14.7 ±0.4 260 ±8 15.5 ±0.6 Gln87Tyr- Enz 311 ±2 17.6 ±0.2 333 ±5 18.1 ±0.9 x2rp Enz-Inh 269 ±4 19.6 ±0.3 288 ±6 20.7 ± 0.5 d) +10 ions +11 ions Protein or Complex Maximum H / D exchange level Saturation time (min) Maximum H / D exchange level Saturation time (min) Asn44Ala 268 ±4 6.8 ±0.2 202 ±8 7.6 ±0.2 Asn44Ala- Enz 348 ±8 6.5 ±0.4 310±2 9.3 ±0.3 X 2 D N J Enz-Inh 299 ±2 7.2 ±0.5 291 ±2 10.9 ±0.6 Asn44Ala- Enz 308 ±7 11.2 ±0.6 337 ±2 13.1 ±0.7 X 2 I L Enz-Inh 269 ±2 11.8 ± 0.4 260 ±8 15.5 ±0.6 Asn44Ala- Enz 320 ±2 11.9 ±0.5 333 ±5 18.1 ±0.9 X 2 I F Enz-Inh 266 ±3 17.0 ±0.3 288 ±6 20.7 ± 0.5 Table 5.3 Maximum H / D exchange levels and saturation times for the +10 and +11 charge states of a) WT Cex, b) Gln76Met, c) Gln87Tyr and d) Asn44Ala and their complexes. 4 0 0 3 5 0 3 0 0 2 5 0 2 0 0 a) 5 150 100 + 1 0 £ n z + -\0Enzlnh — i — 18 — i — 12 10  14 16 S a t u r a t i o n T i m e (min) 20 22 4 U U 350 3 0 0 2 5 0 200 150 100 b) • + 11 Enz + 11 Enzlnh 10 12 14 S a t u r a t i o n T i m e (m in 16 18 20 22 Figure 5.16 Maximum H/D exchange level vs. saturation time for a) +10 and b) +11 charge states, of Enz and Enz • Inh ions of all complexes. show that protein ions from solutions with proteins only, have lower levels of the exchange than the ions from the solutions of complexes. This finding is not consistent with others and needs further investigation. Figures 5.16 a) and b) show plots of the maximum H/D exchange levels for the +10 and +11 charge states, respectively, of Enz and Enz • Inh ions of all the complexes vs. the saturation times. Figures 5.17 a) and b) show plots of the maximum H/D exchange level vs. AGjissociation of the complexes in solution for the +10 and +11 charge states, respectively. It can be seen that there is no correlation between exchange level and saturation time or AGjissociatiOH, suggesting that the solution conformation or flexibility does not depend on the type of the complex. 82 400 _ 350 CD > CD 300 250 200 150 0.20 0.25 0.30 0.35 A G ° dissociation <eV) a) • +10 Enz • +10 Enzlnh 0.40 0.45 400 350 300 250 200 150 100 5-1 — 0.35 dissociation (®^) b) + 11 Enz + 11 Enzlnh 0.20 0.25 0.30 AG0 0.40 0.45 Figure 5.17 Maximum H/D exchange level vs. AG0dissociation in the solution for a) +10 and b) +11 charge states, of all the complexes. 83 5.6.2. Comparison of AEint in the Gas Phase with AGdissociation in Solution Figure 5.18 shows values of AE i n t plotted against the values of A G 0 for dissociation of the complexes in solution. In solution, the inhibition strength increases in the order: X 2 DNJ < X 2 IL < X 2IF for all the proteins. As can be seen from Table 5.1 and Figure 5.18, the AE i n t values for all the ions of the protein complexes follow the same trends. From Figure 5.18, it is evident that for each inhibitor the internal energy needed for dissociation in the gas phase increases in the order Asn44Ala < Gln87Tyr < Gln87Met < WT Cex, for both charge states, the same as the order of increase of A G 0 for dissociation in solution. These same changes in binding are seen irrespective of the identity of the proximal azasugar. Thus binding to the proximal sugar in the gas phase complex appears to be similar to the binding in solution. Mutations that remove or alter individual hydrogen bonds to the distal xylose to reduce the solution binding strength, also reduce the energy required to cause dissociation in the gas phase ions. Therefore individual hydrogen bonds involved in interactions with Gln87 and Asn44 in solution probably remain in the gas phase. Individual hydrogen bonds between heme and myoglobin were previously shown to persist in the gas phase holoprotein [48]. It is likely that the solution structures are partially preserved in the gas phase in these protein small-molecule complexes in part because inhibitor binding stabilizes the complex, holding the binding pocket together. 84 160 -a) 140 - Gln87Met Gln87Tyr^^-^ / / 120 -X 2 I L 100 -Asn44Ala / Gln87Tyr v j £ Gln87Met r 80 -Gln87Tyr Gln87Met WT 60 -Asn44Ala / 40 -X 2 DNJ i Asn44Ala 1 i i 0.20 0.25 0.30 0.35 0.40 0.45 A G ° d / s s o c / a , o n (eV) in solut ion 160 - b) WT G l n 8 7 M e t ^ X2IF 140 -Gln87Tyr 120 -100 - Asn44Ala / X 2 I L Iwr j j ^ - — W T 80 -Gln.S7.Vk-i Gln87Mel 60 - Gln87Tyr Asn44AJa o /1 yr 40 - X 2 DNJ Asri44Ala 0.20 0.25 0.30 0.35 0.40 0.45 ^dissociation (eV) i n S O l u t i ° n Figure 5.18 Internal energies needed for dissociation of ions of the complexes of WT, Gln87Met, Gln87Tyr and Asn44Ala with inhibitors in the gas phase vs. A G 0 for dissociation of the complexes in solution for charge states a) +10 and b) +11. 85 Kitova et al. reported activation energies for dissociation of trisaccharides from an antibody single chain fragment measured in BIRD experiments, and found that activation energies did not correlate with change in standard enthalpy AH0 for association of the complexes in solution. This was attributed to structural differences between the solution and gas phase proteins. If such differences exist in the Cex complexes, they do not alter the order of the binding energies in the gas phase. It is possible the difference in time scales between these MS/MS experiments and the BIRD experiments contributes to these different results. Ions are formed and dissociate in tens of microseconds in the triple quadrupole experiments. In the BIRD experiments, theions are trapped for times of a few to more than a hundred seconds. On this longer time scale the ions may undergo unfolding or refolding transitions to structures that differ markedly from the solution structures [128]. 86 Chapter 6 Conclusions and Recommendations for Future Work The results presented in this thesis represent a detailed study of binding in gas phase protein-ligand complexes. Tandem mass spectrometry results show that the additional energies required to cause dissociation of ions of the complexes in the gas phase, AEint, show a strong correlation with the change in the standard free energy of dissociation, &G~dhsociation' m solution. This suggests that much of the solution binding pocket is preserved in the gas phase. The correlation between collision cross sections and dissociation energies suggests that binding of the ligand helps stabilization of the complex in the gas phase. Conformations are tighter after ligand binding, since the protein folds around the ligand. This is shown through collision cross section measurements in the gas phase. Solution conformation studies by hydrogen/deuterium exchange show that the protein ions form solutions of complexes have somewhat more compact conformations than ions of complexes from the same solution. 87 It is shown that mutant versions of the protein can be used to probe the effects of removal of individual sets of interactions in the complex. It is also shown that tandem mass spectrometry can be used to detect changes of a single hydrogen bond in a complex. The mutant proteins and WT Cex differ by a single amino acid and single hydrogen bond between the protein and the inhibitor. The observed differences in the internal energies needed to cause the dissociation of inhibitors from gas phase complexes with these proteins show that the change in a single amino acid can be detected by tandem mass spectrometry. In addition, by mass spectrometry it is possible to detect the small differences in the binding strengths in the solution, although a comparison of absolute energies is not possible. Overall, this thesis contributes to the significant collections of publications that explore binding in gas phase noncovalent complexes. The method used here, combining tandem mass spectrometry with systematic alterations to the binding partners, should be applicable to a broad range of noncovalent complexes. Investigation of different types of noncovalent complexes by this method is needed to confirm its applicability. Once the results are obtained it should be clear if the solvent has an effect on binding and if the dissociation energies for the complexes in the gas phase follow the same trend as binding energies in the solution. It can subsequently be speculated which type of noncovalent complexes are suitable for study by mass spectrometry and how that depends on their structure, function or nature. 88 This project could be continued in several directions. First, the method used here can be used to study binding of Bex, a xylanase from microorganism Bacillus circulans, and a member of family 11 xylanases. Cex and Bex, are not only related enzymes, but also have similar inhibitors. Therefore, it would be useful to examine if the small differences in the binding between two families of enzymes can be detected by mass spectrometry. Second, gas phase H/D exchange experiments could be done with Cex complexes, to complement the results of H/D exchange in the solution. This would provide another useful comparison of the gas phase conformations to the conformations of the complexes in the solution. 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