1 Protein Structure: Current and Future Directions ............. 1
Shannon M. Swiatkowski and Mark R. Chance
1.1 Introduction ............................................ 1
1.2 Hydrogen/Deuterium Exchange Mass Spectrometry ........... 3
1.3 Hydroxyl-Radical-Mediated Protein Footprinting .......... 5
1.4 Chemical Cross-linking .................................. 8
References ................................................... 8
2 Hydrogen Exchange Mass Spectrometry: Principles and
Capabilities ................................................ 11
Sibastien Brier and John R. Engen
2.1 The Chemistry of Hydrogen Exchange ..................... 11
2.1.1 Principles of Proton Transfer ................... 11
2.1.2 Mechanisms of Backbone Amide Hydrogen
Exchange ........................................ 14
2.1.3 Factors Affecting Hydrogen Exchange ............. 15
2.2 HX Mechanisms in Proteins .............................. 19
2.3 Deuterium Incorporation into Proteins .................. 23
2.3.1 Continuous Labeling ............................. 23
2.3.2 Pulse Labeling .................................. 25
2.3.3 Other Labeling Strategies ....................... 26
2.4 Measuring HX with Mass Spectrometry .................... 26
2.4.1 Global Versus Local Exchange .................... 27
2.4.2 Back Exchange ................................... 27
2.4.3 Proteolysis Before MS ........................... 28
2.4.4 Mass Measurements and Data Processing ........... 29
2.5 Capabilities of HX MS in Structural Biology ............ 31
2.5.1 Protein Folding Studies ......................... 32
2.5.2 Quality Control ................................. 32
2.5.3 Aid in Structure Elucidation .................... 34
2.5.4 Interactions and Dynamics ....................... 35
Acknowledgment .............................................. 36
References .................................................. 36
3 Covalent Labeling Methods for Examining Protein Structure
and Protein Interactions .................................... 45
Keiji Takamoto and Janna Kiselar
3.1 Introduction ........................................... 45
3.2 Chemistry of Hydroxyl Radical Footprinting ............. 46
3.2.1 Generation of Hydroxyl Radicals ................. 47
3.2.2 Reactions of Hydroxyl Radical Products: Nature
of Amino Acid Modifications ..................... 48
3.2.3 Relative Reactivity of Amino Acid Side Chains ... 49
3.2.4 Principles of Hydroxyl Footprinting and
Protein Integrity During Radiolysis ............. 50
3.3 Mass Spectrometry Approaches for Quantitative Protein
Footprinting ........................................... 52
3.3.1 Quantification of Peptide Oxidation Using
LC-MS ........................................... 52
3.3.2 Confirmation of Peptide Identity and
Determination of Modification Sites by MS/MS .... 54
3.4 Examples of Various Methods that Generate Hydroxyl
Radicals in Solution to Examine Protein Structure ...... 55
3.4.1 Radiolytic Footprinting of Cytochrome с ......... 55
3.4.2 Fenton Hydroxyl Radical Footprinting ............ 56
3.4.3 Laser Photolysis of H2O2 ........................ 57
3.4.4 Radiolysis by High-Voltage Electric Discharge
Within ESI Ion Source ........................... 57
3.4.5 Synchrotron X-Ray Footprinting for Protein
Complexes and Assembly Studies: Probing Arp2/3
Complex Activation by ATP and WASp Binding
Proteins ........................................ 58
3.5 The Future: Hybrid Approaches that Combine
Experimental and Computational Data .................... 62
References .................................................. 63
4 Complementary Methods for Structure Determination:
Hydroxyl-Radical-Mediated Footprinting and Deuterium
Exchange Mass Spectrometry as Applied to Serpin Structure ... 69
Xiaojing Zheng and Patrick L. Wintrode
4.1 Introduction ........................................... 69
4.2 Technical Comparison of Hydroxyl-Radical-Mediated
Footprinting and H/D Exchange Methodologies ............ 73
4.3 Structural Mass Spectrometry Data ...................... 76
4.3.1 H/D Exchange Data ............................... 76
4.3.2 Synchrotron Footprinting Data ................... 76
4.4 Solvent Accessibility .................................. 79
4.5 Dynamics ............................................... 84
4.6 Significance for Serpin Structure and Function ......... 87
4.7 Summary ................................................ 87
Acknowledgment .............................................. 88
References .................................................. 88
5 Deuterium Exchange Approaches for Examining Protein
Interactions: Case Studies of Complex Formation ............. 91
Elizabeth A. Komives
5.1 Interactions of Regulatory and Catalytic Subunits
of Protein Kinase A .................................... 91
5.1.1 Interaction of the Catalytic Subunit with a
Pseudosubstrate ................................. 92
5.1.2 Interaction of the Catalytic Domain with the
Riα Regulatory Domain .......................... 92
5.1.3 Combination Of H/D Exchange Data and
Computational Docking ........................... 93
5.2 Allostery in Protein-Protein Interactions Revealed
by H/D Exchange ........................................ 95
5.2.1 Allostery within the Regulatory Subunit
Revealed by H/D Exchange ........................ 95
5.2.2 Allostery in the Thrombin-Thrombomodulin
Interaction ..................................... 97
5.3 Interactions of the Inhibitor Ikα with the
Transcription Factor NF-kB ............................. 97
5.3.1 H/D Exchange of IkBα Reveals Partially
Unfolded Regions ................................ 98
5.3.2 H/D Exchange Reveals IкВα Folds upon Binding
to NF-kB ........................................ 98
References ................................................. 101
6 Hydrogen/Deuterium Exchange Studies of Viruses ............. 105
Sebyung Rang and Peter E. Prevelige Jr.
6.1 Overview of Virus Lifecycles .......................... 105
6.2 Structural Investigations of Viral Capsids ............ 105
6.3 Dynamics of Viral Capsids ............................. 106
6.4 Hydrogen/Deuterium Exchange Studies of Virus Capsid
Structure ............................................. 107
6.4.1 Bacteriophage P22 .............................. 107
6.4.2 HIV ............................................ 109
6.4.3 Brome Mosaic Virus ............................. 113
6.5 Hydrogen/Deuterium Exchange Studies of Viral Protein
Dynamics .............................................. 114
6.5.1 Bacteriophage Phi-29 Scaffolding Protein ....... 114
6.5.2 Packaging Motor P4 from dsRNA Bacteriophages
Phi-8 and Phi-12 ............................... 117
6.6 Technical Aspects of Performing Hydrogen/Deuterium
Exchange Experiments on Viruses ........................ 118
6.6.1 Dissociation of Structures ..................... 118
6.6.2 Presence of Nucleic Acid ....................... 118
6.6.3 Potential for Strain Variation ................. 119
6.6.4 Presence of BSA ................................ 119
6.6.5 Complexity and Size ............................ 119
References ................................................. 119
7 Use of Enhanced Peptide Amide Hydrogen/Deuterium
Exchange-Mass Spectrometry (DXMS) in the Examination
of Protein-Protein Interactions ............................ 123
Yoshitomo Hamuro, Stephen J. Coales, Lora L. Hamuro, and
Virgil L. Woods Jr.
7.1 Introduction .......................................... 123
7.2 Theory of H/D Exchange ................................ 124
7.2.1 Amide H/D Exchange ............................. 124
7.2.2 Protection Factor .............................. 125
7.2.3 Backbone Amide Hydrogens as Thermodynamic
Sensors ........................................ 125
7.2.4 H/D Exchange for Protein-Protein
Interactions ................................... 126
7.3 Overview of DXMS Technology for Protein-Protein
Interactions .......................................... 126
7.3.1 On-Exchange Reaction ........................... 126
7.3.2 Quench of Exchange Reaction .................... 127
7.3.3 Protein Fragmentation by Proteolysis ........... 127
7.3.4 HPLC Separation ................................ 128
7.3.5 Mass Analysis .................................. 128
7.3.6 DXMS of a Protein with or without Protein
Binding Partner ................................ 128
7.4 DXMS of Human Growth Hormone and Its Binding
Protein ............................................... 129
7.4.1 Human Growth Hormone High Affinity Variant ..... 129
7.4.2 DXMS Experiments of Human Growth Hormone and
Its Binding Protein ............................ 130
7.4.3 DXMS of hGHwt and hGHv without hGHbp ........... 131
7.4.4 DXMS of hGHwt and hGHv with hGHbp .............. 131
7.4.5 DXMS of hGHbp with or without hGH .............. 132
7.4.6 Enhanced Affinity by Increasing the Free
Energy of the Unbound State .................... 133
7.5 DXMS of PKA Regulatory Subunits ....................... 133
7.5.1 Protein Kinase A (PKA) Regulatory Subunits ..... 133
7.5.2 DXMS Experiments of PKA Regulatory Subunits .... 134
7.5.3 DXMS of cAMP-Bound PKA R-Subunits .............. 134
7.5.4 Interaction between R-Subunits and C-Subunit ... 136
7.5.5 Interaction between R-Subunits and cAMP ........ 138
7.5.6 Lack of Significant Effects on the D/D Domain
upon Binding to cAMP or C-Subunit .............. 139
7.6 DXMS of PKA R-Subunit D/D Domains and D-AKAP2 AKB
Domain ................................................ 139
7.6.1 PKA R-Subunit D/D Domains and D-AKAP2 AKB
Domain ......................................... 139
7.6.2 DXMS Experiments of PKA R-Subunit D/D Domains
and D-AKAP2 AKB Domain ......................... 140
7.6.3 DXMS of D-AKAP2 AKB Domain with or without
PKA R-Subunit D/D Domains ...................... 141
7.6.4 DXMS of PKA R-Subunit D/D Domains with or
without D-AKAP2 AKB Domain ..................... 143
7.7 Epitope Mapping by DXMS ............................... 146
7.7.1 Epitope Mapping ................................ 146
7.7.2 DXMS Experiments of Cytochrome с in the
Presence and Absence of Antibody ............... 147
7.7.3 Antibody Binding Site on Cytochrome с by
DXMS ........................................... 147
7.7.4 Comparison with X-ray Crystallographic
Structure ...................................... 147
7.8 Conclusions ........................................... 148
Abbreviations .............................................. 149
Acknowledgments ............................................ 150
References ................................................. 150
8 Cross-linking as a Tool to Examine Protein Complexes:
Examples of Cross-linking Strategies and Computational
Modeling ................................................... 157
Evgeniy V. Petrotchenko and Christoph H. Borchers
8.1 Introduction .......................................... 157
8.2 Cross-linking Strategies .............................. 157
8.3 Cross-linking Methodology ............................. 158
8.4 Challenges Associated with Combining Cross-linking
with Mass Spectrometry ................................ 159
8.5 Advances in Mass Spectrometry Instrumentation and
Capabilities .......................................... 160
8.6 Novel Cross-linking Reagents for Mass Spectrometry
Applications .......................................... 162
8.7 Analytical Software ................................... 165
8.8 Using Cross-linking Distance Constraints to Build
Experimental Models of Protein Complexes .............. 167
References ................................................. 167
9 Complex Formation in the Actin Cytoskeleton: Cross-
linking Tools to Define Actin Protein Structure and
Interactions ............................................... 169
Sabrina Benchaar and Emil Reisler
9.1 Introduction .......................................... 169
9.2 Mapping Cross-linking with Methods Other than Mass
Spectrometry .......................................... 171
9.3 Actin-Actin Cross-linking ............................. 171
9.3.1 Intermolecular Cross-linking in F-Actin by N,
N'-p-Phenylene-Dimaleimide between
Lysine 191 and Cysteine 374 .................... 171
9.3.2 Intermolecular Cross-linking in F-Actin by
N-(4-Azidobenzoyl)-Putrescine between
Glutamine 41 and Lysine 113 .................... 172
9.4 Intrastrand Cross-linked Actin between GLN41 and
CYS374 ................................................ 174
9.5 Regulation of Cytoskeleton by ABPs and Mapping their
Interfaces with Actin by Cross-linking ................ 175
9.5.1 Actin-Depolymerizing Factor/Cofilin ............ 175
9.5.2 Mapping the Interaction of Cofilin with
Subdomain 2 on G-Actin ......................... 176
9.5.3 Cofilin-Induced Switch from Intramolecular to
Intermolecular Cross-linking in Skeletal
F-Actin ........................................ 178
9.5.4 The Main Cofilin Binding Site on G-Actin ....... 178
9.6 Cross-linking of Actin and Muscle Proteins - Examples
of Experimental Approaches ............................ 181
9.7 Concluding Remarks .................................... 182
Acknowledgment ............................................. 183
References ................................................. 183
10 Computational Approaches to Examining Protein-Protein
Interactions: Combining Experimental and Computational
Data in the Era of Structural Genomics ..................... 189
J.K. Amisha Kamal
10.1 Interactome in Structural Genomics .................... 189
10.2 Importance of Computational Methods in Structural
Genomics .............................................. 190
10.3 Combining Computational Method with Experimental
Data in Modeling the Structure of Protein Binary
Complex ............................................... 190
10.3.1 General Strategy of the Method ................. 191
10.3.2 Docking Complexes of Known Crystal Structures
without Using Footprinting Constraints ......... 192
10.3.3 Radiolytic Footprinting: G-Actin/GSl and G-
Actin/Cofilin .................................. 198
10.3.4 Docking Complex of Known Crystal Structure
Using Footprinting Constraints ................. 201
10.3.5 Docking Complex of Unknown Crystal Structure
Using Footprinting Constraints ................. 205
10.4 Method Summary ........................................ 208
10.5 Experimental Methods .................................. 210
10.5.1 Radiolysis ..................................... 210
10.5.2 Mass Spectrometry .............................. 210
10.5.3 Solvent-Accessible Surface Area Calculation .... 210
10.5.4 Homology Modeling .............................. 211
10.5.5 Protein-Protein Docking ........................ 211
10.5.6 Docking with Experimental Constraints .......... 211
10.5.7 Electrostatic Potential Surface Mapping and
Calculation of Interface Parameters ............ 212
10.5.8 Footprinting Interface Consistency Score ....... 212
Acknowledgment ............................................. 212
References ................................................. 213
11 Studies of Intact Proteins and Protein Complexes: ESI MS
Approaches ................................................. 217
Igor A. Kaltashov, Rinat R. Abzalimov, Agya K. Frimpong,
and Stephen J. Eyles
11.1 Introduction .......................................... 217
11.2 Tertiary Structure Integrity and Conformational
Heterogeneity (Charge State Distributions) ............ 220
11.3 Quaternary Structure Integrity and Composition of
Non-Covalent Complexes ................................ 224
11.4 Functional Competence ................................. 226
11.5 Flexibility Maps and Binding Interfaces ............... 229
11.6 Gas Phase Ion Chemistry and Its Influence on the
Measurement of Protein Properties in Solution ......... 231
11.7 Challenges and Future Outlook ......................... 234
Acknowledgments ............................................ 237
References ................................................. 237
12 Two Approaches to Mass Spectrometric Protein
Footprinting: PLIMSTEX and FPOP ............................ 243
Michael L. Gross, Mei M. Zhu, and David M. Hambly
12.1 Introduction: Protein-Ligand Interactions by Mass
Spectrometry, Titration, and Hydrogen/Deuterium
Amide Exchange and Fast Photochemical Oxidation of
Proteins .............................................. 243
12.2 Protein-Ligand Interactions by Mass Spectrometry,
Titration, and H/D Amide Exchange (PLIMSTEX) .......... 245
12.2.1 General Protocol for PLIMSTEX .................. 245
12.2.2 Titration Curves ............................... 245
12.3 Applications of PLIMSTEX .............................. 247
12.3.1 Determination of Association Constant (Ka),
Stoichiometry (n), and Protection (ΔDi) ........ 247
12.3.2 Ras-GDP Interacting with Mg2+: A 1:1 Protein:
Metal Ion Interaction .......................... 247
12.3.3 The Interactions of Apo-Calmodulin with Ca2+:
A 1:4 Protein: Metal Ion Interaction ........... 249
12.3.4 Applications in Biologically Relevant Media .... 250
12.3.5 The Interaction of Holo-CaM and Peptides ....... 251
12.4 Self-Association of Insulin: A Protein/Protein
Interaction ........................................... 253
12.5 Features of PLIMSTEX .................................. 254
12.6 Fast Photochemical Oxidation of Proteins: An Example
of Fast Protein Footprinting .......................... 256
12.6.1 Hydroxyl Radicals as a Probe ................... 256
12.6.2 Fast Hydroxyl-Radical Footprinting ............. 258
12.6.3 Locating the Sites of Radical Reaction ......... 258
12.6.4 Application of FPOP to Apomyoglobin ............ 259
12.7 Features of FPOP ...................................... 263
12.8 Future ................................................ 264
Abbreviations .............................................. 265
Acknowledgments ............................................ 265
References ................................................. 265
Index ......................................................... 271
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