Foreword, Professor Sir Alan Fersht ............................ xv
Preface ...................................................... xvii
About the Author .............................................. xix
Acknowledgments ............................................... xxi
Abbreviations and Acronyms .................................. xxiii
1 Principles of Protein Structure and Function ................. 1
1.1 Physical Forces That Shape Protein Structure ............ 1
1.2 Primary Structure: Amino Acid Sequence .................. 3
1.3 Protein-Coding Genes .................................... 4
1.4 Post-Translational Modifications of Amino Acids ......... 5
1.5 Hierarchical Description of Structure ................... 6
1.5.1 Secondary Structure .............................. 6
1.5.2 Tertiary Structure ............................... 9
1.5.3 Quaternary Structure ............................ 11
1.6 Folding of a Protein ................................... 12
1.6.1 Thermodynamic Aspects of Protein Folding ........ 12
1.6.2 Kinetic Aspects of Protein Folding .............. 13
1.6.3 Mechanism of Protein Folding .................... 14
1.6.4 Folding and Chaperones .......................... 14
1.7 Unfolding of a Protein: Lessons from Polymer Theory .... 15
1.8 The Limits of Global Descriptions of the Unfolded
State .................................................. 17
1.9 Databases of Proteins and Protein Structures ........... 17
1.10 DisProt: The Database of Disordered Proteins ........... 18
1.11 The Classical Structure-Function Paradiqm .............. 18
2 A Brief History of Protein Disorder ......................... 21
2.1 Can We Define Disorder? ................................ 21
2.2 The History of Disorder ................................ 22
2.2.1 The Legacy of the Lock-and-Key Hypothesis ....... 22
2.2.2 Structural Adaptability of Binding Sites ........ 23
2.2.3 Polymer Theory and Protein Folding .............. 23
2.2.4 Caseins Are Different ........................... 24
2.2.5 If a Protein Does Not Crystallize ............... 24
2.2.6 The Advent of NMR ............................... 25
2.3 So We Have Disordered Proteins ......................... 27
3 Indirect Techniques for Recognizing and Characterizing
Protein Disorder ............................................ 31
3.1 Resistance to Heat ..................................... 31
3.2 Resistance to Chemical Denaturation .................... 33
3.3 Unusual SDS-PAGE Mobility .............................. 33
3.4 Enhanced Proteolytic Sensitivity ....................... 34
3.5 Limited Proteolysis and Local Structure ................ 35
3.6 Differential Scanning Calorimetry ...................... 35
3.6.1 Transition to a More Ordered State .............. 37
3.6.2 Residual Structure in Calpastatin ............... 37
3.7 Isothermal Titration Calorimetry ....................... 37
3.7.1 The Energetics of Binding of a PPII Helix to
Its Cognate SH3 Domain .......................... 38
3.7.2 Binding of the KID Domain of p27Kip1 to Cyciin
A-Cdk2 .......................................... 38
3.8 Chemical Cross-Linking ................................. 40
3.9 H/D Exchange ........................................... 41
4 Hydrodynamic Techniques ..................................... 43
4.1 Gel Filtration (Size-Exclusion) Chromatography ......... 43
4.2 Dynamic Light Scattering ............................... 45
4.3 Analytical Ultracentrifugation ......................... 46
4.4 Small-Angle X-Ray Scattering ........................... 47
4.4.1 Measles Virus Nucleoprotein ..................... 50
4.4.2 Bacterial Cellulase ............................. 51
4.4.3 p53 ............................................. 52
4.5 Pulsed-Field Gradient NMR .............................. 53
5 Spectroscopic Techniques for Characterizing Disorder ........ 55
5.1 X-Ray Crystallography .................................. 55
5.2 Fluorescence Spectroscopy .............................. 57
5.2.1 UV Fluorescence ................................. 58
5.2.2 Fluorescence Quenching .......................... 58
5.2.3 ANS Binding ..................................... 60
5.2.4 Fluorescence Resonance Energy Transfer .......... 60
5.2.5 Fluorescence Correlation Spectroscopy ........... 61
5.2.5.1 Dimensions of an IDP and the Effect
of Crowding ............................ 61
5.2.5.2 Internal Protein Dynamics .............. 62
5.3 Fourier-Transform Infrared Resonance Spectroscopy ...... 62
5.4 Circular Dichroism ..................................... 63
5.5 Raman Optical Activity Spectroscopy .................... 65
5.6 Electron Paramagnetic Resonance Spectroscopy ........... 66
5.7 Electron Microscopy .................................... 69
5.8 Atomic Force Microscopy ................................ 71
5.8.1 Matrix Metalloproteinase 9 ...................... 72
5.8.2 α-Synuclein ..................................... 72
6 Nuclear Magnetic Resonance .................................. 73
6.1 Basic Principles ....................................... 73
6.2 Global Characterization by NMR ......................... 74
6.2.1 1-D1HNMR ........................................ 74
6.2.2 Wide-Line NMR ................................... 75
6.2.3 Pulsed-Field Gradient NMR ....................... 76
6.2.4 HSQC ............................................ 76
6.3 Sequence-Specific Structural Information ............... 78
6.3.1 Chemical Shifts ................................. 79
6.3.2 Dynamic Information from Relaxation Data ........ 79
6.3.3 Distance Information from NOE ................... 81
6.3.4 Coupling Constants .............................. 82
6.4 Special Applications ................................... 83
6.4.1 Combinations with MD ............................ 83
6.4.2 Amide Proton Exchange Rate ...................... 83
6.4.3 In-Cell NMR ..................................... 84
7 Proteomic Approaches for the Identification of IDPs ......... 85
7.1 Expectations and Limitations of Proteomic Studies ...... 85
7.2 2DE-MS Identification of Proteins in Extracts
Enriched for Disorder .................................. 86
7.3 Native/Urea 2DE Provides Direct Information on
Disorder ............................................... 88
8 IDPs under Conditions Approaching In Vivo ................... 91
8.1 Macromolecular Crowding in the Cell .................... 91
8.2 In Vitro Approaches to Mimicking Crowding Conditions ... 92
8.3 The State of IDPs In Vivo .............................. 95
8.3.1 Proteasomal Degradation ......................... 95
8.3.2 In-Cell NMR ..................................... 97
8.4 Physiological Half-Life of IDPs: No Signs of Rapid
Degradation ............................................ 99
8.5 Indirect Considerations Underscoring Disorder of
IDPs In Vivo .......................................... 100
9 Prediction of Disorder ..................................... 103
9.1 General Points ........................................ 103
9.2 Propensity-Based Predictors ........................... 103
9.2.1 Prediction of Low-Complexity Regions ........... 106
9.2.2 Charge-Hydropathy Plot ......................... 106
9.2.3 Prediction of Globularity and Disorder ......... 107
9.2.4 Composition and Hydrophobic Cluster Analysis ... 108
9.3 Machine-Learning Algorithms ........................... 109
9.3.1 Neural Networks ................................ 109
9.3.2 Support-Vector Machines ........................ 110
9.4 Prediction Based on Interresidue Contacts ............. 111
9.4.1 Contact Numbers of Amino Acids ................. 111
9.4.2 Estimating Pair-Wise Interresidue Interaction
Energies ....................................... 112
9.4.3 Predictor of Contact Potentials ................ 112
9.5 Prediction of Short and Long Regions of Disorder
Separately ............................................ 112
9.6 Combination of Predictors: Meta-Servers ............... 114
9.7 Prediction of Functional Motifs In IDPs ............... 115
9.8 Comparison of the Accuracy of Predictors: The CASP
Experiment ............................................ 116
9.9 A Better Target Prioritization in Structural
Genomics .............................................. 119
10 Structure of IDPs .......................................... 121
10.1 Primary Structure of Disordered Proteins .............. 121
10.1.1 Amino Acid Composition ......................... 121
10.1.2 Sequence Features Characterizing Disorder ...... 123
10.1.3 Flavors of Disorder? ........................... 124
10.2 Secondary Structure of Disordered Proteins ............ 125
10.2.1 Secondary Structure in Solution State: Signs
of Transient Order ............................. 125
10.2.2 A Lot of PPII Helix Conformation ............... 126
10.2.3 Secondary Structure in Solution State:
Sequence-Specific Information .................. 127
10.2.3.1 p27Kip1 ................................ 127
10.2.3.2 CREBKID ............................... 130
10.2.3.3 Tau Protein ........................... 131
10.2.3.4 Fibronectin-Binding Protein A ......... 131
10.2.3.5 α-Synuclein ........................... 132
10.2.3.6 p53 ................................... 132
10.2.3.7 Calpastatin ........................... 132
10.2.4 Secondary Structure in the Bound State ......... 133
10.3 Ambiguity in Structure ................................ 134
10.3.1 Chameleon Sequences ............................ 134
10.3.2 Dual-Personality Sequences ..................... 135
10.3.3 The Twilight Zone between Order and Disorder ... 135
10.4 Tertiary Structure: Global Features of IDP
Structures ............................................ 136
10.4.1 Hydrodynamic Description ....................... 136
10.4.2 Spectroscopic Approaches ....................... 137
10.4.3 Global Structure: Is It Related to the
Structure in the Bound State? .................. 139
10.5 Dynamics of IDP Structure: The Time-Course of
Fluctuations within the Ensemble ...................... 140
10.5.1 The Importance of Dynamics in Structural
Descriptions ................................... 140
10.5.1.1 Local/Segmental Motions ............... 140
10.5.1.2 Restricted Segmental Motions .......... 141
10.5.1.3 Reduced Local Motion Signals
Transient Structural Elements ......... 141
10.5.2 A Reduction in Motility Signals Disorder-to-
Order Transition ................................. 142
10.6 A Readout of Structure: The Hydrate Layer of IDPs ..... 142
11 Biological Processes Enriched in Disorder .................. 143
11.1 Biological Functions Enriched in Disorder ............. 143
11.2 Disorder in Transcription/Transcription Regulation .... 145
11.2.1 Transcription Factors .......................... 145
11.2.2 Transcription Co-Activators .................... 146
11.2.3 Disorder in the Core Apparatus ................. 148
11.3 Disorder in Signaling Proteins ........................ 149
11.3.1 Receptors and Membrane Proteins ................ 149
11.3.2 Scaffold Proteins and Hub Proteins ............. 151
11.3.3 Regulation of the Cell Cycle ................... 152
11.4 Nucleic Acid-Containing Organells ..................... 152
11.4.1 Ribosome ....................................... 152
11.4.2 Disorder in Chromatin Organization ............. 153
11.4.2.1 Histones .............................. 153
11.4.2.2 Other Chromatin Organizing Proteins ... 154
11.5 Disorder in RNA-Binding Proteins: Transcription and
RNA Folding ........................................... 155
11.6 Cytoskeletal Proteins ................................. 157
11.6.1 Microfilaments ................................. 157
11.6.2 Intermediate Filaments ......................... 158
11.6.3 Microtubules ................................... 159
11.7 Disorder in Stress Proteins ........................... 159
11.8 Disorder and Metal Binding ............................ 160
11.9 Disorder and Enzyme Activity .......................... 161
11.10 Is There a Link between the Pattern of Disorder and
Function? ............................................. 162
12 Molecular Functions of Disordered Proteins ................. 163
12.1 Entropic Chain Functions .............................. 163
12.1.1 Linkers and Spacers ............................ 163
12.1.2 Entropic Clocks ................................ 165
12.1.3 Entropic Springs ............................... 166
12.1.4 Entropic Bristles/Brushes ...................... 166
12.2 Display Site Functions ................................ 168
12.2.1 Phosphorylation Sites .......................... 168
12.2.2 Sites of Proteolytic Processing ................ 170
12.2.3 Ubiquitination Sites ........................... 171
12.2.4 Acetylation Sites .............................. 172
12.3 Chaperone Functions ................................... 172
12.3.1 Disorder in Protein Chaperones ................. 174
12.3.2 Disorder in RNA Chaperones ..................... 174
12.4 Effector Functions .................................... 176
12.4.1 Inhibitors ..................................... 177
12.4.2 Activators ..................................... 177
12.5 Scavenger Functions ................................... 178
12.5.1 Salivary Proline-Rich Glycoproteins ............ 178
12.5.2 Caseins ........................................ 178
12.5.3 Calsequestrin .................................. 179
12.6 Assembler Functions ................................... 179
12.6.1 Targeting Activity ............................. 179
12.6.2 Assembling Complexes ........................... 180
12.6.2.1 HMGA, a Fully Disordered Hub
Protein ............................... 183
12.6.2.2 MDM2, a Partially Disordered Hub
Protein ............................... 183
12.6.2.3 Calmodulin, an Ordered Hub Protein .... 184
12.6.2.4 Disorder and Complex Size ............. 185
12.6.2.5 Scaffold Proteins ..................... 185
12.7 Prion Functions ....................................... 187
12.7.1 Sup35 .......................................... 187
12.7.2 Cytoplasmic Polyadenylation Element Binding
Protein ........................................ 188
13 Evolution and Prevalence of Disorder ....................... 189
13.1 Phylogenetic Distribution of Disorder ................. 189
13.1.1 Predicted Disorder in Genomes and Proteomes .... 189
13.1.2 The Origin of Disordered Proteins in
Eukaryotes ..................................... 191
13.1.3 The Generation of Disordered Domains by
Gene Duplication and Module Exchange ........... 192
13.2 Fast Evolution of IDPs by Point Mutations ............. 193
13.2.1 Neutrality in the Evolution of IDPs ............ 194
13.2.2 Disordered Regions May Also Be Conserved ....... 195
13.3 Fast Evolution of IDPs by Repeat Expansion ............ 195
13.3.1 Micro- and Minisatellites in Protein
Evolution ...................................... 196
13.3.1.1 Mechanisms of Repeat Expansion ........ 197
13.3.1.2 Tandem Repeats in the CTD of RNA
Polymerase II ......................... 198
13.3.1.3 Tandem Repeats in the PEVK
Region of Titin ....................... 198
13.3.1.4 Tandem Repeats in Prion Protein ....... 199
13.3.2 A Functional Model of Repeat Expansion in
IDPs ........................................... 199
13.4 Fast Evolution and Functionality of Disordered
Proteins .............................................. 200
13.4.1 Retention of Entropic-Chain Functions and
Recognition Functions .......................... 200
13.4.2 Recognition Another Way: The Lessons from
Fuzziness ...................................... 202
13.4.3 Co-Evolution of IDPs and Their Partners ........ 202
13.5 Structural Variability and Evolvability of New
Functions ............................................. 203
14 Extension of the Structure-Function Paradigm ............... 205
14.1 Functions That Stem Directly from the Disordered
State ................................................. 205
14.2 Recognition Functions: Recognition by Short Motifs .... 206
14.2.1 Preformed Structural Elements .................. 206
14.2.2 Linear Motifs .................................. 208
14.2.3 Molecular Recognition Elements/Features ........ 210
14.2.4 Recognition by Domain-Sized Motifs and
Mutual Folding ................................. 210
14.2.5 Recognition Interfaces ......................... 212
14.2.6 Unification of Concepts? ....................... 214
14.3 Disorder-to-Order Transition in Recognition:
Mechanistic and Thermodynamic Aspects ................. 214
14.3.1 Site-Directed Mutagenesis Studies of Induced
Folding ........................................ 215
14.3.2 Molecular Dynamics Simulations of Induced
Folding ........................................ 216
14.3.3 NMR Studies of the Mechanism of Induced
Folding ........................................ 217
14.3.4 The Analogy of Folding and Induced Folding ..... 218
14.4 Recognition Functions: Uncoupling Specificity from
Binding Strength ...................................... 219
14.4.1 Disorder May Contribute to Recognition of
Specific Sites ................................. 219
14.4.2 Disorder May Make Interactions Weaker .......... 220
14.4.3 Strong Multivalent Binding and Weak Aspecific
Binding ........................................ 220
14.5 Implications of Disorder for the Kinetics of
Interactions .......................................... 221
14.5.1 Primary Contact Sites .......................... 222
14.5.2 Fly-Casting in Recognition ..................... 222
14.6 Adaptability and Moonlighting ......................... 223
14.7 Nested Interfaces ..................................... 225
14.8 Disorder in the Bound State: Fuzziness ................ 226
14.8.1 Structural Polymorphism in the Bound State ..... 226
14.8.2 Clamp-Type of Fuzziness ........................ 227
14.8.3 Flanking-Type of Fuzziness ..................... 228
14.8.4 Random-Type of Fuzziness ....................... 228
14.9 Processivity of Binding ............................... 229
14.10 Sequence Independence In Recognition ................. 230
14.11 Ultrasensitivity of Recognition ...................... 230
14.11.1 Recognition of Sic1 by Cdc4 ................... 231
14.11.2 Regulation of CFTR by Its Disordered R
Domain ........................................ 231
14.11.3 Electrostatics in Ultrasensitivity ............ 232
14.12 Signal Propagation in the Structural Ensemble of
IDPs .................................................. 232
14.12.1 The Signaling Conduit P27kip1 .................. 232
14.12.2 Tailored Auto-Activation of WASP .............. 233
14.12.3 Allostery Mediated by Order-Disorder
Transitions ................................... 234
14.13 Disorder and Alternative Splicing .................... 234
14.14 Molecular Mimicry by a Disordered Region ............. 235
14.15 Entropy Transfer in Chaperone Action ................. 235
15 Structural Disorder and Disease ............................ 237
15.1 Structural Disorder and Cancer ........................ 237
15.1.1 Disorder in Cancer-Associated Proteins ......... 237
15.1.2 P53 ............................................ 238
15.1.3 Cip/Kip Cdk Inhibitors ......................... 240
15.1.4 Breast-Cancer 1 ................................ 242
15.1.5 Securin (PTTG) ................................. 243
15.1.6 Disorder in Proteins Generated by Chromosomal
Translocations ................................. 244
15.2 Structural Disorder in Proteins Involved in
Cardiovascular Diseases, Diabetes, and Autoimmune
Diseases .............................................. 245
15.3 Structural Disorder and Neurodegenerative Diseases .... 246
15.3.1 Alzheimer's Disease ............................ 248
15.3.1.1 A p Peptide ........................... 248
15.3.1.2 Таu Protein ........................... 248
15.3.2 Parkinson's Disease ............................ 249
15.3.2.1 α-Synuclein (NACP) .................... 250
15.3.3 Glutamine-Repeat Diseases ...................... 251
15.3.3.1 Huntington's Disease .................. 252
15.3.3.2 Huntingtin ............................ 253
15.3.4 Prion Diseases ................................. 253
15.3.4.1 Prion Protein ......................... 254
15.4 Systemic Amyloidoses .................................. 255
15.5 Common Themes in Amyloid Formation .................... 255
15.5.1 Kinetics of Amyloid Formation .................. 256
15.5.2 Disorder in Amyloidogenic Proteins ............. 256
15.5.3 The Structure of the Amyloid ................... 257
15.5.4 Molecular Mechanism of Transition to the
Amyloid State .................................. 258
15.6 Does Structural Disorder Pose a Danger? ............... 259
15.7 Disorder in Pathogenic Organisms ...................... 260
15.8 Rational Drug Design Based on Protein Disorder ........ 262
15.6 References ............................................ 265
Index ......................................................... 313
|
