Bücher | Chemie | Protein Folding Handbook | Inhaltsverzeichnis

Bücher Bitte auswählen Anthropologie Architektur Archäologie Bauwesen Bildungswesen Biomedizintechnik Biowissenschaften Chemie Chemische Verfahrenstechnik Elektrotechnik Finanzwesen Fischforschung Fremdsprachen Für Kinder Gastronomie Geographie Geowissenschaft Geschichte Gesundheit Grafikdesign Humanistische Studien Industrielle Verfahrenstechnik Informatik IT für Anwender Kommunikation Krankenpflege Kulturwissenschaften Kunst und Angewandte Kunst Landwirtschaft Lebensmittelforschung Lifestyle Literaturwissenschaft Maschinenbau Materialwissenschaften Mathematik Medizin Nachschlagewerke Nanotechnologie Naturwissenschaft/Technik Pferdeforschung Philosophie Physik Politikwissenschaft Polymerwissenschaft Prüfungsvorbereitung Psychologie Ratgeber Rechnungswesen Rechtswissenschaft Reisen Religion Sozialpolitik Soziologie Sprachwissenschaften Statistik Umweltforschung Veterinärmedizin Volkswirtschaftslehre Wirtschaft Zahnmedizin   Soeben erschienen Titelsuche Featured Sites Unterhaltung Zeitschriften Elektronische Medien Bitte auswählen Anthropologie Architektur Archäologie Bauwesen Bildungswesen Biomedizintechnik Biowissenschaften Chemie Chemische Verfahrenstechnik Elektrotechnik Finanzwesen Fischforschung Fremdsprachen Für Kinder Gastronomie Geographie Geowissenschaft Geschichte Gesundheit Grafikdesign Humanistische Studien Industrielle Verfahrenstechnik Informatik IT für Anwender Kommunikation Krankenpflege Kulturwissenschaften Kunst und Angewandte Kunst Landwirtschaft Lebensmittelforschung Lifestyle Literaturwissenschaft Maschinenbau Materialwissenschaften Mathematik Medizin Nachschlagewerke Nanotechnologie Naturwissenschaft/Technik Pferdeforschung Philosophie Physik Politikwissenschaft Polymerwissenschaft Prüfungsvorbereitung Psychologie Ratgeber Rechnungswesen Rechtswissenschaft Reisen Religion Sozialpolitik Soziologie Sprachwissenschaften Statistik Umweltforschung Veterinärmedizin Volkswirtschaftslehre Wirtschaft Zahnmedizin   Contents     Part I, Volume 1   Preface LVIII Contributors of Part I LX I/1 Principles of Protein Stability and Design 1 1 Early Days of Studying the Mechanism of Protein Folding Robert L. Baldwin 3 1.1 Introduction 3 1.2 Two-state Folding 4 1.3 Levinthal's Paradox 5 1.4 The Domain as a Unit of Folding 6 1.5 Detection of Folding Intermediates and Initial Work on the Kinetic Mechanism of Folding 7 1.6 Two Unfolded Forms of RNase A and Explanation by Proline Isomerization 9 1.7 Covalent Intermediates in the Coupled Processes of Disulfide Bond Formation and Folding 11 1.8 Early Stages of Folding Detected by Antibodies and by Hydrogen Exchange 12 1.9 Molten Globule Folding Intermediates 14 1.10 Structures of Peptide Models for Folding Intermediates 15 Acknowledgments 16 References 16 2 Spectroscopic Techniques to Study Protein Folding and Stability Franz Schmid 22 2.1 Introduction 22 2.2 Absorbance 23 2.2.1 Absorbance of Proteins 23 2.2.2 Practical Considerations for the Measurement of Protein Absorbance 27 2.2.3 Data Interpretation 29 2.3 Fluorescence 29 2.3.1 The Fluorescence of Proteins 30 2.3.2 Energy Transfer and Fluorescence Quenching in a Protein: Barnase 31 2.3.3 Protein Unfolding Monitored by Fluorescence 33 2.3.4 Environmental Effects on Tyrosine and Tryptophan Emission 36 2.3.5 Practical Considerations 37 2.4 Circular Dichroism 38 2.4.1 CD Spectra of Native and Unfolded Proteins 38 2.4.2 Measurement of Circular Dichroism 41 2.4.3 Evaluation of CD Data 42 References 43 3 Denaturation of Proteins by Urea and Guanidine Hydrochloride C. Nick Pace, Gerald R. Grimsley, and J. Martin Scholtz 45 3.1 Historical Perspective 45 3.2 How Urea Denatures Proteins 45 3.3 Linear Extrapolation Method 48 3.4 G(H2O) 50 3.5 m-Values 55 3.6 Concluding Remarks 58 3.7 Experimental Protocols 59 3.7.1 How to Choose the Best Denaturant for your Study 59 3.7.2 How to Prepare Denaturant Solutions 59 3.7.3 How to Determine Solvent Denaturation Curves 60 3.7.3.1 Determining a Urea or GdmCl Denaturation Curve 62 3.7.3.2 How to Analyze Urea or GdmCl Denaturant Curves 63 3.7.4 Determining Differences in Stability 64 Acknowledgments 65 References 65 4 Thermal Unfolding of Proteins Studied by Calorimetry George I. Makhatadze 70 4.1 Introduction 70 4.2 Two-state Unfolding 71 4.3 Cold Denaturation 76 4.4 Mechanisms of Thermostabilization 77 4.5 Thermodynamic Dissection of Forces Contributing to Protein Stability 79 4.5.1 Heat Capacity Changes, Cp} 81 4.5.2 Enthalpy of Unfolding, H 81 4.5.3 Entropy of Unfolding, S 83 4.6 Multistate Transitions 84 4.6.1 Two-state Dimeric Model 85 4.6.2 Two-state Multimeric Model 86 4.6.3 Three-state Dimeric Model 86 4.6.4 Two-state Model with Ligand Binding 88 4.6.5 Four-state (Two-domain Protein) Model 90 4.7 Experimental Protocols 92 4.7.1 How to Prepare for DSC Experiments 92 4.7.2 How to Choose Appropriate Conditions 94 4.7.3 Critical Factors in Running DSC Experiments 94 References 95 5 Pressure--Temperature Phase Diagrams of Proteins Wolfgang Doster and Josef Friedrich 99 5.1 Introduction 99 5.2 Basic Aspects of Phase Diagrams of Proteins and Early Experiments 100 5.3 Thermodynamics of Pressure--Temperature Phase Diagrams 103 5.4 Measuring Phase Stability Boundaries with Optical Techniques 110 5.4.1 Fluorescence Experiments with Cytochrome c 110 5.4.2 Results 112 5.5 What Do We Learn from the Stability Diagram? 116 5.5.1 Thermodynamics 116 5.5.2 Determination of the Equilibrium Constant of Denaturation 117 5.5.3 Microscopic Aspects 120 5.5.4 Structural Features of the Pressure-denatured State 122 5.6 Conclusions and Outlook 123 Acknowledgment 124 References 124 6 Weak Interactions in Protein Folding: Hydrophobic Free Energy, van der Waals Interactions, Peptide Hydrogen Bonds, and Peptide Solvation Robert L. Baldwin 127 6.1 Introduction 127 6.2 Hydrophobic Free Energy, Burial of Nonpolar Surface and van der Waals Interactions 128 6.2.1 History 128 6.2.2 Liquid--Liquid Transfer Model 128 6.2.3 Relation between Hydrophobic Free Energy and Molecular Surface Area 130 6.2.4 Quasi-experimental Estimates of the Work of Making a Cavity in Water or in Liquid Alkane 131 6.2.5 Molecular Dynamics Simulations of the Work of Making Cavities in Water 133 6.2.6 Dependence of Transfer Free Energy on the Volume of the Solute 134 6.2.7 Molecular Nature of Hydrophobic Free Energy 136 6.2.8 Simulation of Hydrophobic Clusters 137 6.2.9 Cp and the Temperature-dependent Thermodynamics of Hydrophobic Free Energy 137 6.2.10 Modeling Formation of the Hydrophobic Core from Solvation Free Energy and van der Waals Interactions between Nonpolar Residues 142 6.2.11 Evidence Supporting a Role for van der Waals Interactions in Forming the Hydrophobic Core 144 6.3 Peptide Solvation and the Peptide Hydrogen Bond 145 6.3.1 History 145 6.3.2 Solvation Free Energies of Amides 147 6.3.3 Test of the Hydrogen-Bond Inventory 149 6.3.4 The Born Equation 150 6.3.5 Prediction of Solvation Free Energies of Polar Molecules by an Electrostatic Algorithm 150 6.3.6 Prediction of the Solvation Free Energies of Peptide Groups in Different Backbone Conformations 151 6.3.7 Predicted Desolvation Penalty for Burial of a Peptide H-bond 153 6.3.8 Gas--Liquid Transfer Model 154 Acknowledgments 156 References 156 7 Electrostatics of Proteins: Principles, Models and Applications Sonja Braun-Sand and Arieh Warshel 163 7.1 Introduction 163 7.2 Historical Perspectives 163 7.3 Electrostatic Models: From Microscopic to Macroscopic Models 166 7.3.1 All-Atom Models 166 7.3.2 Dipolar Lattice Models and the PDLD Approach 168 7.3.3 The PDLD/S-LRA Model 170 7.3.4 Continuum (Poisson-Boltzmann) and Related Approaches 171 7.3.5 Effective Dielectric Constant for Charge--Charge Interactions and the GB Model 172 7.4 The Meaning and Use of the Protein Dielectric Constant 173 7.5 Validation Studies 176 7.6 Systems Studied 178 7.6.1 Solvation Energies of Small Molecules 178 7.6.2 Calculation of pKa Values of Ionizable Residues 179 7.6.3 Redox and Electron Transport Processes 180 7.6.4 Ligand Binding 181 7.6.5 Enzyme Catalysis 182 7.6.6 Ion Pairs 183 7.6.7 Protein--Protein Interactions 184 7.6.8 Ion Channels 185 7.6.9 Helix Macrodipoles versus Localized Molecular Dipoles 185 7.6.10 Folding and Stability 186 7.7 Concluding Remarks 189 Acknowledgments 190 References 190 8 Protein Conformational Transitions as Seen from the Solvent: Magnetic Relaxation Dispersion Studies of Water, Co-solvent, and Denaturant Interactions with Nonnative Proteins Bertil Halle, Vladimir P. Denisov, Kristofer Modig, and Monika Davidovic 201 8.1 The Role of the Solvent in Protein Folding and Stability 201 8.2 Information Content of Magnetic Relaxation Dispersion 202 8.3 Thermal Perturbations 205 8.3.1 Heat Denaturation 205 8.3.2 Cold Denaturation 209 8.4 Electrostatic Perturbations 213 8.5 Solvent Perturbations 218 8.5.1 Denaturation Induced by Urea 219 8.5.2 Denaturation Induced by Guanidinium Chloride 225 8.5.3 Conformational Transitions Induced by Co-solvents 228 8.6 Outlook 233 8.7 Experimental Protocols and Data Analysis 233 8.7.1 Experimental Methodology 233 8.7.1.1 Multiple-field MRD 234 8.7.1.2 Field-cycling MRD 234 8.7.1.3 Choice of Nuclear Isotope 235 8.7.2 Data Analysis 236 8.7.2.1 Exchange Averaging 236 8.7.2.2 Spectral Density Function 237 8.7.2.3 Residence Time 239 8.7.2.4 19F Relaxation 240 8.7.2.5 Coexisting Protein Species 241 8.7.2.6 Preferential Solvation 241 References 242 9 Stability and Design of -Helices Andrew J. Doig, Neil Errington, and Teuku M. Iqbalsyah 247 9.1 Introduction 247 9.2 Structure of the -Helix 247 9.2.1 Capping Motifs 248 9.2.2 Metal Binding 250 9.2.3 The 310-Helix 251 9.2.4 The -Helix 251 9.3 Design of Peptide Helices 252 9.3.1 Host--Guest Studies 253 9.3.2 Helix Lengths 253 9.3.3 The Helix Dipole 253 9.3.4 Acetylation and Amidation 254 9.3.5 Side Chain Spacings 255 9.3.6 Solubility 256 9.3.7 Concentration Determination 257 9.3.8 Design of Peptides to Measure Helix Parameters 257 9.3.9 Helix Templates 259 9.3.10 Design of 310-Helices 259 9.3.11 Design of -helices 261 9.4 Helix Coil Theory 261 9.4.1 Zimm-Bragg Model 261 9.4.2 Lifson-Roig Model 262 9.4.3 The Unfolded State and Polyproline II Helix 265 9.4.4 Single Sequence Approximation 265 9.4.5 N- and C-Caps 266 9.4.6 Capping Boxes 266 9.4.7 Side-chain Interactions 266 9.4.8 N1, N2, and N3 Preferences 267 9.4.9 Helix Dipole 267 9.4.10 310- and -Helices 268 9.4.11 AGADIR 268 9.4.12 Lomize-Mosberg Model 269 9.4.13 Extension of the Zimm-Bragg Model 270 9.4.14 Availability of Helix/Coil Programs 270 9.5 Forces Affecting -Helix Stability 270 9.5.1 Helix Interior 270 9.5.2 Caps 273 9.5.3 Phosphorylation 276 9.5.4 Noncovalent Side-chain Interactions 276 9.5.5 Covalent Side-chain interactions 277 9.5.6 Capping Motifs 277 9.5.7 Ionic Strength 279 9.5.8 Temperature 279 9.5.9 Trifluoroethanol 279 9.5.10 pKa Values 280 9.5.11 Relevance to Proteins 281 9.6 Experimental Protocols and Strategies 281 9.6.1 Solid Phase Peptide Synthesis (SPPS) Based on the Fmoc Strategy 281 9.6.1.1 Equipment and Reagents 281 9.6.1.2 Fmoc Deprotection and Coupling 283 9.6.1.3 Kaiser Test 284 9.6.1.4 Acetylation and Cleavage 285 9.6.1.5 Peptide Precipitation 286 9.6.2 Peptide Purification 286 9.6.2.1 Equipment and Reagents 286 9.6.2.2 Method 286 9.6.3 Circular Dichroism 287 9.6.4 Acquisition of Spectra 288 9.6.4.1 Instrumental Considerations 288 9.6.5 Data Manipulation and Analysis 289 9.6.5.1 Protocol for CD Measurement of Helix Content 291 9.6.6 Aggregation Test for Helical Peptides 291 9.6.6.1 Equipment and Reagents 291 9.6.6.2 Method 292 9.6.7 Vibrational Circular Dichroism 292 9.6.8 NMR Spectroscopy 292 9.6.8.1 Nuclear Overhauser Effect 293 9.6.8.2 Amide Proton Exchange Rates 294 9.6.8.3 13C NMR 294 9.6.9 Fourier Transform Infrared Spectroscopy 295 9.6.9.1 Secondary Structure 295 9.6.10 Raman Spectroscopy and Raman Optical Activity 296 9.6.11 pH Titrations 298 9.6.11.1 Equipment and Reagents 298 9.6.11.2 Method 298 Acknowledgments 299 References 299 10 Design and Stability of Peptide -Sheets Mark S. Searle 314 10.1 Introduction 314 10.2 -Hairpins Derived from Native Protein Sequences 315 10.3 Role of -Turns in Nucleating -Hairpin Folding 316 10.4 Intrinsic , Propensities of Amino Acids 319 10.5 Side-chain Interactions and -Hairpin Stability 321 10.5.1 Aromatic Clusters Stabilize -Hairpins 322 10.5.2 Salt Bridges Enhance Hairpin Stability 325 10.6 Cooperative Interactions in -Sheet Peptides: Kinetic Barriers to Folding 330 10.7 Quantitative Analysis of Peptide Folding 331 10.8 Thermodynamics of -Hairpin Folding 332 10.9 Multistranded Antiparallel -Sheet Peptides 334 10.10 Concluding Remarks: Weak Interactions and Stabilization of Peptide -Sheets 339 References 340 11 Predicting Free Energy Changes of Mutations in Proteins Raphael Guerois, Joaquim Mendes, and Luis Serrano 343 11.1 Physical Forces that Determine Protein Conformational Stability 343 11.1.1 Protein Conformational Stability [1] 343 11.1.2 Structures of the N and D States [2--6] 344 11.1.3 Studies Aimed at Understanding the Physical Forces that Determine Protein Conformational Stability [1, 2, 8, 19--26] 346 11.1.4 Forces Determining Conformational Stability [1, 2, 8, 19--27] 346 11.1.5 Intramolecular Interactions 347 11.1.5.1 van der Waals Interactions 347 11.1.5.2 Electrostatic Interactions 347 11.1.5.3 Conformational Strain 349 11.1.6 Solvation 350 11.1.7 Intramolecular Interactions and Solvation Taken Together 350 11.1.8 Entropy 351 11.1.9 Cavity Formation 352 11.1.10 Summary 353 11.2 Methods for the Prediction of the Effect of Point Mutations on in vitro Protein Stability 353 11.2.1 General Considerations on Protein Plasticity upon Mutation 353 11.2.2 Predictive Strategies 355 11.2.3 Methods 356 11.2.3.1 From Sequence and Multiple Sequence Alignment Analysis 356 11.2.3.2 Statistical Analysis of the Structure Databases 356 11.2.3.3 Helix/Coil Transition Model 357 11.2.3.4 Physicochemical Method Based on Protein Engineering Experiments 359 11.2.3.5 Methods Based only on the Basic Principles of Physics and Thermodynamics 364 11.3 Mutation Effects on in vivo Stability 366 11.3.1 The N-terminal Rule 366 11.3.2 The C-terminal Rule 367 11.3.3 PEST Signals 368 11.4 Mutation Effects on Aggregation 368 References 369 I/2 Dynamics and Mechanisms of Protein Folding Reactions 377 12.1 Kinetic Mechanisms in Protein Folding Annett Bachmann and Thomas Kiefhaber 379 12.1.1 Introduction 379 12.1.2 Analysis of Protein Folding Reactions using Simple Kinetic Models 379 12.1.2.1 General Treatment of Kinetic Data 380 12.1.2.2 Two-state Protein Folding 380 12.1.2.3 Complex Folding Kinetics 384 12.1.2.3.1 Heterogeneity in the Unfolded State 384 12.1.2.3.2 Folding through Intermediates 388 12.1.2.3.3 Rapid Pre-equilibria 391 12.1.2.3.4 Folding through an On-pathway High-energy Intermediate 393 12.1.3 A Case Study: the Mechanism of Lysozyme Folding 394 12.1.3.1 Lysozyme Folding at pH 5.2 and Low Salt Concentrations 394 12.1.3.2 Lysozyme Folding at pH 9.2 or at High Salt Concentrations 398 12.1.4 Non-exponential Kinetics 401 12.1.5 Conclusions and Outlook 401 12.1.6 Protocols -- Analytical Solutions of Three-state Protein Folding Models 402 12.1.6.1 Triangular Mechanism 402 12.1.6.2 On-pathway Intermediate 403 12.1.6.3 Off-pathway Mechanism 404 12.1.6.4 Folding Through an On-pathway High-Energy Intermediate 404 Acknowledgments 406 References 406 12.2 Characterization of Protein Folding Barriers with Rate Equilibrium Free Energy Relationships Thomas Kiefhaber, Ignacio E. Sánchez, and Annett Bachmann 411 12.2.1 Introduction 411 12.2.2 Rate Equilibrium Free Energy Relationships 411 12.2.2.1 Linear Rate Equilibrium Free Energy Relationships in Protein Folding 414 12.2.2.2 Properties of Protein Folding Transition States Derived from Linear REFERs 418 12.2.3 Nonlinear Rate Equilibrium Free Energy Relationships in Protein Folding 420 12.2.3.1 Self-Interaction and Cross-Interaction Parameters 420 12.2.3.2 Hammond and Anti-Hammond Behavior 424 12.2.3.3 Sequential and Parallel Transition States 425 12.2.3.4 Ground State Effects 428 12.2.4 Experimental Results on the Shape of Free Energy Barriers in Protein Folding 432 12.2.4.1 Broadness of Free Energy Barriers 432 12.2.4.2 Parallel Pathways 437 12.2.5 Folding in the Absence of Enthalpy Barriers 438 12.2.6 Conclusions and Outlook 438 Acknowledgments 439 References 439 13 A Guide to Measuring and Interpreting -values Nicholas R. Guydosh and Alan R. Fersht 445 13.1 Introduction 445 13.2 Basic Concept of -Value Analysis 445 13.3 Further Interpretation of 448 13.4 Techniques 450 13.5 Conclusions 452 References 452 14 Fast Relaxation Methods Martin Gruebele 454 14.1 Introduction 454 14.2 Techniques 455 14.2.1 Fast Pressure-Jump Experiments 455 14.2.2 Fast Resistive Heating Experiments 456 14.2.3 Fast Laser-induced Relaxation Experiments 457 14.2.3.1 Laser Photolysis 457 14.2.3.2 Electrochemical Jumps 458 14.2.3.3 Laser-induced pH Jumps 458 14.2.3.4 Covalent Bond Dissociation 459 14.2.3.5 Chromophore Excitation 460 14.2.3.6 Laser Temperature Jumps 460 14.2.4 Multichannel Detection Techniques for Relaxation Studies 461 14.2.4.1 Small Angle X-ray Scattering or Light Scattering 462 14.2.4.2 Direct Absorption Techniques 463 14.2.4.3 Circular Dichroism and Optical Rotatory Dispersion 464 14.2.4.4 Raman and Resonance Raman Scattering 464 14.2.4.5 Intrinsic Fluorescence 465 14.2.4.6 Extrinsic Fluorescence 465 14.3 Protein Folding by Relaxation 466 14.3.1 Transition State Theory, Energy Landscapes, and Fast Folding 466 14.3.2 Viscosity Dependence of Folding Motions 470 14.3.3 Resolving Burst Phases 471 14.3.4 Fast Folding and Unfolded Proteins 472 14.3.5 Experiment and Simulation 472 14.4 Summary 474 14.5 Experimental Protocols 475 14.5.1 Design Criteria for Laser Temperature Jumps 475 14.5.2 Design Criteria for Fast Single-Shot Detection Systems 476 14.5.3 Designing Proteins for Fast Relaxation Experiments 477 14.5.4 Linear Kinetic, Nonlinear Kinetic, and Generalized Kinetic Analysis of Fast Relaxation 477 14.5.4.1 The Reaction D F in the Presence of a Barrier 477 14.5.4.2 The Reaction 2A A2 in the Presence of a Barrier 478 14.5.4.3 The Reaction D F at Short Times or over Low Barriers 479 14.5.5 Relaxation Data Analysis by Linear Decomposition 480 14.5.5.1 Singular Value Decomposition (SVD) 480 14.5.5.2 -Analysis 481 Acknowledgments 481 References 482 15 Early Events in Protein Folding Explored by Rapid Mixing Methods Heinrich Roder, Kosuke Maki, Ramil F. Latypov, Hong Cheng, and M. C. Ramachandra Shastry 491 15.1 Importance of Kinetics for Understanding Protein Folding 491 15.2 Burst-phase Signals in Stopped-flow Experiments 492 15.3 Turbulent Mixing 494 15.4 Detection Methods 495 15.4.1 Tryptophan Fluorescence 495 15.4.2 ANS Fluorescence 498 15.4.3 FRET 499 15.4.4 Continuous-flow Absorbance 501 15.4.5 Other Detection Methods used in Ultrafast Folding Studies 502 15.5 A Quenched-Flow Method for H-D Exchange Labeling Studies on the Microsecond Time Scale 502 15.6 Evidence for Accumulation of Early Folding Intermediates in Small Proteins 505 15.6.1 B1 Domain of Protein G 505 15.6.2 Ubiquitin 508 15.6.3 Cytochrome c 512 15.7 Significance of Early Folding Events 515 15.7.1 Barrier-limited Folding vs. Chain Diffusion 515 15.7.2 Chain Compaction: Random Collapse vs. Specific Folding 516 15.7.3 Kinetic Role of Early Folding Intermediates 517 15.7.4 Broader Implications 520 Appendix 521 A1 Design and Calibration of Rapid Mixing Instruments 521 A1.1 Stopped-flow Equipment 521 A1.2 Continuous-flow Instrumentation 524 Acknowledgments 528 References 528 16 Kinetic Protein Folding Studies using NMR Spectroscopy Markus Zeeb and Jochen Balbach 536 16.1 Introduction 536 16.2 Following Slow Protein Folding Reactions in Real Time 538 16.3 Two-dimensional Real-time NMR Spectroscopy 545 16.4 Dynamic and Spin Relaxation NMR for Quantifying Microsecond-to-Millisecond Folding Rates 550 16.5 Conclusions and Future Directions 555 16.6 Experimental Protocols 556 16.6.1 How to Record and Analyze 1D Real-time NMR Spectra 556 16.6.1.1 Acquisition 556 16.6.1.2 Processing 557 16.6.1.3 Analysis 557 16.6.1.4 Analysis of 1D Real-time Diffusion Experiments 558 16.6.2 How to Extract Folding Rates from 1D Spectra by Line Shape Analysis 559 16.6.2.1 Acquisition 560 16.6.2.2 Processing 560 16.6.2.3 Analysis 561 16.6.3 How to Extract Folding Rates from 2D Real-time NMR Spectra 562 16.6.3.1 Acquisition 563 16.6.3.2 Processing 563 16.6.3.3 Analysis 563 16.6.4 How to Analyze Heteronuclear NMR Relaxation and Exchange Data 565 16.6.4.1 Acquisition 566 16.6.4.2 Processing 567 16.6.4.3 Analysis 567 Acknowledgments 569 References 569 Part I, Volume 2   17 Fluorescence Resonance Energy Transfer (FRET) and Single Molecule Fluorescence Detection Studies of the Mechanism of Protein Folding and Unfolding Elisha Haas 573 Abbreviations 573 17.1 Introduction 573 17.2 What are the Main Aspects of the Protein Folding Problem that can be Addressed by Methods Based on FRET Measurements? 574 17.2.1 The Three Protein Folding Problems 574 17.2.1.1 The Chain Entropy Problem 574 17.2.1.2 The Function Problem: Conformational Fluctuations 575 17.3 Theoretical Background 576 17.3.1 Nonradiative Excitation Energy Transfer 576 17.3.2 What is FRET? The Singlet--Singlet Excitation Transfer 577 17.3.3 Rate of Nonradiative Excitation Energy Transfer within a Donor--Acceptor Pair 578 17.3.4 The Orientation Factor 583 17.3.5 How to Determine and Control the Value of Ro? 584 17.3.6 Index of Refraction n 584 17.3.7 The Donor Quantum Yield oD 586 17.3.8 The Spectral Overlap Integral J 586 17.4 Determination of Intramolecular Distances in Protein Molecules using FRET Measurements 586 17.4.1 Single Distance between Donor and Acceptor 587 17.4.1.1 Method 1: Steady State Determination of Decrease of Donor Emission 587 17.4.1.2 Method 2: Acceptor Excitation Spectroscopy 588 17.4.2 Time-resolved Methods 588 17.4.3 Determination of E from Donor Fluorescence Decay Rates 589 17.4.4 Determination of Acceptor Fluorescence Lifetime 589 17.4.5 Determination of Intramolecular Distance Distributions 590 17.4.6 Evaluation of the Effect of Fast Conformational Fluctuations and Determination of Intramolecular Diffusion Coefficients 592 17.5 Experimental Challenges in the Implementation of FRET Folding Experiments 594 17.5.1 Optimized Design and Preparation of Labeled Protein Samples for FRET Folding Experiments 594 17.5.2 Strategies for Site-specific Double Labeling of Proteins 595 17.5.3 Preparation of Double-labeled Mutants Using Engineered Cysteine Residues (strategy 4) 596 17.5.4 Possible Pitfalls Associated with the Preparation of Labeled Protein Samples for FRET Folding Experiments 599 17.6 Experimental Aspects of Folding Studies by Distance Determination Based on FRET Measurements 600 17.6.1 Steady State Determination of Transfer Efficiency 600 17.6.1.1 Donor Emission 600 17.6.1.2 Acceptor Excitation Spectroscopy 601 17.6.2 Time-resolved Measurements 601 17.7 Data Analysis 603 17.7.1 Rigorous Error Analysis 606 17.7.2 Elimination of Systematic Errors 606 17.8 Applications of trFRET for Characterization of Unfolded and Partially Folded Conformations of Globular Proteins under Equilibrium Conditions 607 17.8.1 Bovine Pancreatic Trypsin Inhibitor 607 17.8.2 The Loop Hypothesis 608 17.8.3 RNase A 609 17.8.4 Staphylococcal Nuclease 611 17.9 Unfolding Transition via Continuum of Native-like Forms 611 17.10 The Third Folding Problem: Domain Motions and Conformational Fluctuations of Enzyme Molecules 611 17.11 Single Molecule FRET-detected Folding Experiments 613 17.12 Principles of Applications of Single Molecule FRET Spectroscopy in Folding Studies 615 17.12.1 Design and Analysis of Single Molecule FRET Experiments 615 17.12.1.1 How is Single Molecule FRET Efficiency Determined? 615 17.12.1.2 The Challenge of Extending the Length of the Time Trajectories 617 17.12.2 Distance and Time Resolution of the Single Molecule FRET Folding Experiments 618 17.13 Folding Kinetics 619 17.13.1 Steady State and trFRET-detected Folding Kinetics Experiments 619 17.13.2 Steady State Detection 619 17.13.3 Time-resolved FRET Detection of Rapid Folding Kinetics: the ``Double Kinetics'' Experiment 621 17.13.4 Multiple Probes Analysis of the Folding Transition 622 17.14 Concluding Remarks 625 Acknowledgments 626 References 627 18 Application of Hydrogen Exchange Kinetics to Studies of Protein Folding Kaare Teilum, Birthe B. Kragelund, and Flemming M. Poulsen 634 18.1 Introduction 634 18.2 The Hydrogen Exchange Reaction 638 18.2.1 Calculating the Intrinsic Hydrogen Exchange Rate Constant, kint 638 18.3 Protein Dynamics by Hydrogen Exchange in Native and Denaturing Conditions 641 18.3.1 Mechanisms of Exchange 642 18.3.2 Local Opening and Closing Rates from Hydrogen Exchange Kinetics 642 18.3.2.1 The General Amide Exchange Rate Expression -- the Linderstrøm-Lang Equation 643 18.3.2.2 Limits to the General Rate Expression -- EX1 and EX2 644 18.3.2.3 The Range between the EX1 and EX2 Limits 646 18.3.2.4 Identification of Exchange Limit 646 18.3.2.5 Global Opening and Closing Rates and Protein Folding 647 18.3.3 The ``Native State Hydrogen Exchange'' Strategy 648 18.3.3.1 Localization of Partially Unfolded States, PUFs 650 18.4 Hydrogen Exchange as a Structural Probe in Kinetic Folding Experiments 651 18.4.1 Protein Folding/Hydrogen Exchange Competition 652 18.4.2 Hydrogen Exchange Pulse Labeling 656 18.4.3 Protection Factors in Folding Intermediates 657 18.4.4 Kinetic Intermediate Structures Characterized by Hydrogen Exchange 659 18.5 Experimental Protocols 661 18.5.1 How to Determine Hydrogen Exchange Kinetics at Equilibrium 661 18.5.1.1 Equilibrium Hydrogen Exchange Experiments 661 18.5.1.2 Determination of Segmental Opening and Closing Rates, kop and kcl 662 18.5.1.3 Determination of >Gfluc,m, and Gunf 662 18.5.2 Planning a Hydrogen Exchange Folding Experiment 662 18.5.2.1 Determine a Combination of tpulse and pHpulse 662 18.5.2.2 Setup Quench Flow Apparatus 662 18.5.2.3 Prepare Deuterated Protein and Chemicals 663 18.5.2.4 Prepare Buffers and Unfolded Protein 663 18.5.2.5 Check pH in the Mixing Steps 664 18.5.2.6 Sample Mixing and Preparation 664 18.5.3 Data Analysis 664 Acknowledgments 665 References 665 19 Studying Protein Folding and Aggregation by Laser Light Scattering Klaus Gast and Andreas J. Modler 673 19.1 Introduction 673 19.2 Basic Principles of Laser Light Scattering 674 19.2.1 Light Scattering by Macromolecular Solutions 674 19.2.2 Molecular Parameters Obtained from Static Light Scattering (SLS) 676 19.2.3 Molecular Parameters Obtained from Dynamic Light Scattering (DLS) 678 19.2.4 Advantages of Combined SLS and DLS Experiments 680 19.3 Laser Light Scattering of Proteins in Different Conformational States -- Equilibrium Folding/Unfolding Transitions 680 19.3.1 General Considerations, Hydrodynamic Dimensions in the Natively Folded State 680 19.3.2 Changes in the Hydrodynamic Dimensions during Heat-induced Unfolding 682 19.3.3 Changes in the Hydrodynamic Dimensions upon Cold Denaturation 683 19.3.4 Denaturant-induced Changes of the Hydrodynamic Dimensions 684 19.3.5 Acid-induced Changes of the Hydrodynamic Dimensions 685 19.3.6 Dimensions in Partially Folded States -- Molten Globules and Fluoroalcohol-induced States 686 19.3.7 Comparison of the Dimensions of Proteins in Different Conformational States 687 19.3.8 Scaling Laws for the Native and Highly Unfolded States, Hydrodynamic Modeling 687 19.4 Studying Folding Kinetics by Laser Light Scattering 689 19.4.1 General Considerations, Attainable Time Regions 689 19.4.2 Hydrodynamic Dimensions of the Kinetic Molten Globule of Bovine -Lactalbumin 690 19.4.3 RNase A is Only Weakly Collapsed During the Burst Phase of Folding 691 19.5 Misfolding and Aggregation Studied by Laser Light Scattering 692 19.5.1 Overview: Some Typical Light Scattering Studies of Protein Aggregation 692 19.5.2 Studying Misfolding and Amyloid Formation by Laser Light Scattering 693 19.5.2.1 Overview: Initial States, Critical Oligomers, Protofibrils, Fibrils 693 19.5.2.2 Aggregation Kinetics of A Peptides 694 19.5.2.3 Kinetics of Oligomer and Fibril Formation of PGK and Recombinant Hamster Prion Protein 695 19.5.2.4 Mechanisms of Misfolding and Misassembly, Some General Remarks 698 19.6 Experimental Protocols 698 19.6.1 Laser Light Scattering Instrumentation 698 19.6.1.1 Basic Experimental Set-up, General Requirements 698 19.6.1.2 Supplementary Measurements and Useful Options 700 19.6.1.3 Commercially Available Light Scattering Instrumentation 701 19.6.2 Experimental Protocols for the Determination of Molecular Mass and Stokes Radius of a Protein in a Particular Conformational State 701 Protocol 1 702 Protocol 2 704 Acknowledgments 704 References 704 20 Conformational Properties of Unfolded Proteins Patrick J. Fleming and George D. Rose 710 20.1 Introduction 710 20.1.1 Unfolded vs. Denatured Proteins 710 20.2 Early History 711 20.3 The Random Coil 712 20.3.1 The Random Coil -- Theory 713 20.3.1.1 The Random Coil Model Prompts Three Questions 716 20.3.1.2 The Folding Funnel 716 20.3.1.3 Transition State Theory 717 20.3.1.4 Other Examples 717 20.3.1.5 Implicit Assumptions from the Random Coil Model 718 20.3.2 The Random Coil -- Experiment 718 20.3.2.1 Intrinsic Viscosity 719 20.3.2.2 SAXS and SANS 720 20.4 Questions about the Random Coil Model 721 20.4.1 Questions from Theory 722 20.4.1.1 The Flory Isolated-pair Hypothesis 722 20.4.1.2 Structure vs. Energy Duality 724 20.4.1.3 The ``Rediscovery'' of Polyproline II Conformation 724 20.4.1.4 PII in Unfolded Peptides and Proteins 726 20.4.2 Questions from Experiment 727 20.4.2.1 Residual Structure in Denatured Proteins and Peptides 727 20.4.3 The Reconciliation Problem 728 20.4.4 Organization in the Unfolded State -- the Entropic Conjecture 728 20.4.4.1 Steric Restrictions beyond the Dipeptide 729 20.5 Future Directions 730 Acknowledgments 731 References 731 21 Conformation and Dynamics of Nonnative States of Proteins studied by NMR Spectroscopy Julia Wirmer, Christian Schlörb, and Harald Schwalbe 737 21.1 Introduction 737 21.1.1 Structural Diversity of Polypeptide Chains 737 21.1.2 Intrinsically Unstructured and Natively Unfolded Proteins 739 21.2 Prerequisites: NMR Resonance Assignment 740 21.3 NMR Parameters 744 21.3.1 Chemical shifts 745 21.3.1.1 Conformational Dependence of Chemical Shifts 745 21.3.1.2 Interpretation of Chemical Shifts in the Presence of Conformational Averaging 746 21.3.2 J Coupling Constants 748 21.3.2.1 Conformational Dependence of J Coupling Constants 748 21.3.2.2 Interpretation of J Coupling Constants in the Presence of Conformational Averaging 750 21.3.3 Relaxation: Homonuclear NOEs 750 21.3.3.1 Distance Dependence of Homonuclear NOEs 750 21.3.3.2 Interpretation of Homonuclear NOEs in the Presence of Conformational Averaging 754 21.3.4 Heteronuclear Relaxation (15N R1, R2, hetNOE) 757 21.3.4.1 Correlation Time Dependence of Heteronuclear Relaxation Parameters 757 21.3.4.2 Dependence on Internal Motions of Heteronuclear Relaxation Parameters 759 21.3.5 Residual Dipolar Couplings 760 21.3.5.1 Conformational Dependence of Residual Dipolar Couplings 760 21.3.5.2 Interpretation of Residual Dipolar Couplings in the Presence of Conformational Averaging 763 21.3.6 Diffusion 765 21.3.7 Paramagnetic Spin Labels 766 21.3.8 H/D Exchange 767 21.3.9 Photo-CIDNP 767 21.4 Model for the Random Coil State of a Protein 768 21.5 Nonnative States of Proteins: Examples from Lysozyme, -Lactalbumin, and Ubiquitin 771 21.5.1 Backbone Conformation 772 21.5.1.1 Interpretation of Chemical Shifts 772 21.5.1.2 Interpretation of NOEs 774 21.5.1.3 Interpretation of J Coupling Constants 780 21.5.2 Side-chain Conformation 784 21.5.2.1 Interpretation of J Coupling Constants 784 21.5.3 Backbone Dynamics 786 21.5.3.1 Interpretation of 15N Relaxation Rates 786 21.6 Summary and Outlook 793 Acknowledgments 794 References 794 22 Dynamics of Unfolded Polypeptide Chains Beat Fierz and Thomas Kiefhaber 809 22.1 Introduction 809 22.2 Equilibrium Properties of Chain Molecules 809 22.2.1 The Freely Jointed Chain 810 22.2.2 Chain Stiffness 810 22.2.3 Polypeptide Chains 811 22.2.4 Excluded Volume Effects 812 22.3 Theory of Polymer Dynamics 813 22.3.1 The Langevin Equation 813 22.3.2 Rouse Model and Zimm Model 814 22.3.3 Dynamics of Loop Closure and the Szabo-Schulten-Schulten Theory 815 22.4 Experimental Studies on the Dynamics in Unfolded Polypeptide Chains 816 22.4.1 Experimental Systems for the Study of Intrachain Diffusion 816 22.4.1.1 Early Experimental Studies 816 22.4.1.2 Triplet Transfer and Triplet Quenching Studies 821 22.4.1.3 Fluorescence Quenching 825 22.4.2 Experimental Results on Dynamic Properties of Unfolded Polypeptide Chains 825 22.4.2.1 Kinetics of Intrachain Diffusion 826 22.4.2.2 Effect of Loop Size on the Dynamics in Flexible Polypeptide Chains 826 22.4.2.3 Effect of Amino Acid Sequence on Chain Dynamics 829 22.4.2.4 Effect of the Solvent on Intrachain Diffusion 831 22.4.2.5 Effect of Solvent Viscosity on Intrachain Diffusion 833 22.4.2.6 End-to-end Diffusion vs. Intrachain Diffusion 834 22.4.2.7 Chain Diffusion in Natural Protein Sequences 834 22.5 Implications for Protein Folding Kinetics 837 22.5.1 Rate of Contact Formation during the Earliest Steps in Protein Folding 837 22.5.2 The Speed Limit of Protein Folding vs. the Pre-exponential Factor 839 22.5.3 Contributions of Chain Dynamics to Rate- and Equilibrium Constants for Protein Folding Reactions 840 22.6 Conclusions and Outlook 844 22.7 Experimental Protocols and Instrumentation 844 22.7.1 Properties of the Electron Transfer Probes and Treatment of the Transfer Kinetics 845 22.7.2 Test for Diffusion-controlled Reactions 847 22.7.2.1 Determination of Bimolecular Quenching or Transfer Rate Constants 847 22.7.2.2 Testing the Viscosity Dependence 848 22.7.2.3 Determination of Activation Energy 848 22.7.3 Instrumentation 849 Acknowledgments 849 References 849 23 Equilibrium and Kinetically Observed Molten Globule States Kosuke Maki, Kiyoto Kamagata, and Kunihiro Kuwajima 856 23.1 Introduction 856 23.2 Equilibrium Molten Globule State 858 23.2.1 Structural Characteristics of the Molten Globule State 858 23.2.2 Typical Examples of the Equilibrium Molten Globule State 859 23.2.3 Thermodynamic Properties of the Molten Globule State 860 23.3 The Kinetically Observed Molten Globule State 862 23.3.1 Observation and Identification of the Molten Globule State in Kinetic Refolding 862 23.3.2 Kinetics of Formation of the Early Folding Intermediates 863 23.3.3 Late Folding Intermediates and Structural Diversity 864 23.3.4 Evidence for the On-pathway Folding Intermediate 865 23.4 Two-stage Hierarchical Folding Funnel 866 23.5 Unification of the Folding Mechanism between Non-two-state and Two-state Proteins 867 23.5.1 Statistical Analysis of the Folding Data of Non-two-state and Two-state Proteins 868 23.5.2 A Unified Mechanism of Protein Folding: Hierarchy 870 23.5.3 Hidden Folding Intermediates in Two-state Proteins 871 23.6 Practical Aspects of the Experimental Study of Molten Globules 872 23.6.1 Observation of the Equilibrium Molten Globule State 872 23.6.1.1 Two-state Unfolding Transition 872 23.6.1.2 Multi-state (Three-state) Unfolding Transition 874 23.6.2 Burst-phase Intermediate Accumulated during the Dead Time of Refolding Kinetics 876 23.6.3 Testing the Identity of the Molten Globule State with the Burst-Phase Intermediate 877 References 879 24 Alcohol- and Salt-induced Partially Folded Intermediates Daizo Hamada and Yuji Goto 884 24.1 Introduction 884 24.2 Alcohol-induced Intermediates of Proteins and Peptides 886 24.2.1 Formation of Secondary Structures by Alcohols 888 24.2.2 Alcohol-induced Denaturation of Proteins 888 24.2.3 Formation of Compact Molten Globule States 889 24.2.4 Example: -Lactoglobulin 890 24.3 Mechanism of Alcohol-induced Conformational Change 893 24.4 Effects of Alcohols on Folding Kinetics 896 24.5 Salt-induced Formation of the Intermediate States 899 24.5.1 Acid-denatured Proteins 899 24.5.2 Acid-induced Unfolding and Refolding Transitions 900 24.6 Mechanism of Salt-induced Conformational Change 904 24.7 Generality of the Salt Effects 906 24.8 Conclusion 907 References 908 25 Prolyl Isomerization in Protein Folding Franz Schmid 916 25.1 Introduction 916 25.2 Prolyl Peptide Bonds 917 25.3 Prolyl Isomerizations as Rate-determining Steps of Protein Folding 918 25.3.1 The Discovery of Fast and Slow Refolding Species 918 25.3.2 Detection of Proline-limited Folding Processes 919 25.3.3 Proline-limited Folding Reactions 921 25.3.4 Interrelation between Prolyl Isomerization and Conformational Folding 923 25.4 Examples of Proline-limited Folding Reactions 924 25.4.1 Ribonuclease A 924 25.4.2 Ribonuclease T1 926 25.4.3 The Structure of a Folding Intermediate with an Incorrect Prolyl Isomer 928 25.5 Native-state Prolyl Isomerizations 929 25.6 Nonprolyl Isomerizations in Protein Folding 930 25.7 Catalysis of Protein Folding by Prolyl Isomerases 932 25.7.1 Prolyl Isomerases as Tools for Identifying Proline-limited Folding Steps 932 25.7.2 Specificity of Prolyl Isomerases 933 25.7.3 The Trigger Factor 934 25.7.4 Catalysis of Prolyl Isomerization During de novo Protein Folding 935 25.8 Concluding Remarks 936 25.9 Experimental Protocols 936 25.9.1 Slow Refolding Assays (``Double Jumps'') to Measure Prolyl Isomerizations in an Unfolded Protein 936 25.9.1.1 Guidelines for the Design of Double Jump Experiments 937 25.9.1.2 Formation of US Species after Unfolding of RNase A 938 25.9.2 Slow Unfolding Assays for Detecting and Measuring Prolyl Isomerizations in Refolding 938 25.9.2.1 Practical Considerations 939 25.9.2.2 Kinetics of the Formation of Fully Folded IIHY-G3P* Molecules 939 References 939 26 Folding and Disulfide Formation Margherita Ruoppolo, Piero Pucci, and Gennaro Marino 946 26.1 Chemistry of the Disulfide Bond 946 26.2 Trapping Protein Disulfides 947 26.3 Mass Spectrometric Analysis of Folding Intermediates 948 26.4 Mechanism(s) of Oxidative Folding so Far -- Early and Late Folding Steps 949 26.5 Emerging Concepts from Mass Spectrometric Studies 950 26.5.1 Three-fingered Toxins 951 26.5.2 RNase A 953 26.5.3 Antibody Fragments 955 26.5.4 Human Nerve Growth Factor 956 26.6 Unanswered Questions 956 26.7 Concluding Remarks 957 26.8 Experimental Protocols 957 26.8.1 How to Prepare Folding Solutions 957 26.8.2 How to Carry Out Folding Reactions 958 26.8.3 How to Choose the Best Mass Spectrometric Equipment for Your Study 959 26.8.4 How to Perform Electrospray (ES)MS Analysis 959 26.8.5 How to Perform Matrix-assisted Laser Desorption Ionization (MALDI) MS Analysis 960 References 961 27 Concurrent Association and Folding of Small Oligomeric Proteins Hans Rudolf Bosshard 965 27.1 Introduction 965 27.2 Experimental Methods Used to Follow the Folding of Oligomeric Proteins 966 27.2.1 Equilibrium Methods 966 27.2.2 Kinetic Methods 968 27.3 Dimeric Proteins 969 27.3.1 Two-state Folding of Dimeric Proteins 970 27.3.1.1 Examples of Dimeric Proteins Obeying Two-state Folding 971 27.3.2 Folding of Dimeric Proteins through Intermediate States 978 27.4 Trimeric and Tetrameric Proteins 983 27.5 Concluding Remarks 986 Appendix -- Concurrent Association and Folding of Small Oligomeric Proteins 987 A1 Equilibrium Constants for Two-state Folding 988 A1.1 Homooligomeric Protein 988 A1.2 Heterooligomeric Protein 989 A2 Calculation of Thermodynamic Parameters from Equilibrium Constants 990 A2.1 Basic Thermodynamic Relationships 990 A2.2 Linear Extrapolation of Denaturant Unfolding Curves of Two-state Reaction 990 A2.3 Calculation of the van't Hoff Enthalpy Change from Thermal Unfolding Data 990 A2.4 Calculation of the van't Hoff Enthalpy Change from the Concentration-dependence of Tm 991 A2.5 Extrapolation of Thermodynamic Parameters to Different Temperatures: Gibbs-Helmholtz Equation 991 A3 Kinetics of Reversible Two-state Folding and Unfolding: Integrated Rate Equations 992 A3.1 Two-state Folding of Dimeric Protein 992 A3.2 Two-state Unfolding of Dimeric Protein 992 A3.3 Reversible Two-state Folding and Unfolding 993 A3.3.1 Homodimeric protein 993 A3.3.2 Heterodimeric protein 993 A4 Kinetics of Reversible Two-state Folding: Relaxation after Disturbance of a Pre-existing Equilibrium (Method of Bernasconi) 994 Acknowledgments 995 References 995 28 Folding of Membrane Proteins Lukas K. Tamm and Heedeok Hong 998 28.1 Introduction 998 28.2 Thermodyamics of Residue Partitioning into Lipid Bilayers 1000 28.3 Stability of -Barrel Proteins 1001 28.4 Stability of Helical Membrane Proteins 1009 28.5 Helix and Other Lateral Interactions in Membrane Proteins 1010 28.6 The Membrane Interface as an Important Contributor to Membrane Protein Folding 1012 28.7 Membrane Toxins as Models for Helical Membrane Protein Insertion 1013 28.8 Mechanisms of -Barrel Membrane Protein Folding 1015 28.9 Experimental Protocols 1016 28.9.1 SDS Gel Shift Assay for Heat-modifiable Membrane Proteins 1016 28.9.1.1 Reversible Folding and Unfolding Protocol Using OmpA as an Example 1016 28.9.2 Tryptophan Fluorescence and Time-resolved Distance Determination by Tryptophan Fluorescence Quenching 1018 28.9.2.1 TDFQ Protocol for Monitoring the Translocation of Tryptophans across Membranes 1019 28.9.3 Circular Dichroism Spectroscopy 1020 28.9.4 Fourier Transform Infrared Spectroscopy 1022 28.9.4.1 Protocol for Obtaining Conformation and Orientation of Membrane Proteins and Peptides by Polarized ATR-FTIR Spectroscopy 1023 Acknowledgments 1025 References 1025 29 Protein Folding Catalysis by Pro-domains Philip N. Bryan 1032 29.1 Introduction 1032 29.2 Bimolecular Folding Mechanisms 1033 29.3 Structures of Reactants and Products 1033 29.3.1 Structure of Free SBT 1033 29.3.2 Structure of SBT/Pro-domain Complex 1036 29.3.3 Structure of Free ALP 1037 29.3.4 Structure of the ALP/Pro-domain Complex 1037 29.4 Stability of the Mature Protease 1039 29.4.1 Stability of ALP 1039 29.4.2 Stability of Subtilisin 1040 29.5 Analysis of Pro-domain Binding to the Folded Protease 1042 29.6 Analysis of Folding Steps 1043 29.7 Why are Pro-domains Required for Folding? 1046 29.8 What is the Origin of High Cooperativity? 1047 29.9 How Does the Pro-domain Accelerate Folding? 1048 29.10 Are High Kinetic Stability and Facile Folding Mutually Exclusive? 1049 29.11 Experimental Protocols for Studying SBT Folding 1049 29.11.1 Fermentation and Purification of Active Subtilisin 1049 29.11.2 Fermentation and Purification of Facile-folding Ala221 Subtilisin from E. coli 1050 29.11.3 Mutagenesis and Protein Expression of Pro-domain Mutants 1051 29.11.4 Purification of Pro-domain 1052 29.11.5 Kinetics of Pro-domain Binding to Native SBT 1052 29.11.6 Kinetic Analysis of Pro-domain Facilitated Subtilisin Folding 1052 29.11.6.1 Single Mixing 1052 29.11.6.2 Double Jump: Renaturation--Denaturation 1053 29.11.6.3 Double Jump: Denaturation--Renaturation 1053 29.11.6.4 Triple Jump: Denaturation--Renaturation--Denaturation 1054 References 1054 30 The Thermodynamics and Kinetics of Collagen Folding Hans Peter Ba¨chinger and Ju¨rgen Engel 1059 30.1 Introduction 1059 30.1.1 The Collagen Family 1059 30.1.2 Biosynthesis of Collagens 1060 30.1.3 The Triple Helical Domain in Collagens and Other Proteins 1061 30.1.4 N- and C-Propeptide, Telopeptides, Flanking Coiled-Coil Domains 1061 30.1.5 Why is the Folding of the Triple Helix of Interest? 1061 30.2 Thermodynamics of Collagen Folding 1062 30.2.1 Stability of the Triple Helix 1062 30.2.2 The Role of Posttranslational Modifications 1063 30.2.3 Energies Involved in the Stability of the Triple Helix 1063 30.2.4 Model Peptides Forming the Collagen Triple Helix 1066 30.2.4.1 Type of Peptides 1066 30.2.4.2 The All-or-none Transition of Short Model Peptides 1066 30.2.4.3 Thermodynamic Parameters for Different Model Systems 1069 30.2.4.4 Contribution of Different Tripeptide Units to Stability 1075 30.2.4.5 Crystal and NMR Structures of Triple Helices 1076 30.2.4.6 Conformation of the Randomly Coiled Chains 1077 30.2.4.7 Model Studies with Isomers of Hydroxyproline and Fluoroproline 1078 30.2.4.8 Cis trans Equilibria of Peptide Bonds 1079 30.2.4.9 Interpretations of Stabilities on a Molecular Level 1080 30.3 Kinetics of Triple Helix Formation 1081 30.3.1 Properties of Collagen Triple Helices that Influence Kinetics 1081 30.3.2 Folding of Triple Helices from Single Chains 1082 30.3.2.1 Early Work 1082 30.3.2.2 Concentration Dependence of the Folding of (PPG)10 and (POG)10 1082 30.3.2.3 Model Mechanism of the Folding Kinetics 1085 30.3.2.4 Rate Constants of Nucleation and Propagation 1087 30.3.2.5 Host--guest Peptides and an Alternative Kinetics Model 1088 30.3.3 Triple Helix Formation from Linked Chains 1089 30.3.3.1 The Short N-terminal Triple Helix of Collagen III in Fragment Col1--3 1089 30.3.3.2 Folding of the Central Long Triple Helix of Collagen III 1090 30.3.3.3 The Zipper Model 1092 30.3.4 Designed Collagen Models with Chains Connected by a Disulfide Knot or by Trimerizing Domains 1097 30.3.4.1 Disulfide-linked Model Peptides 1097 30.3.4.2 Model Peptides Linked by a Foldon Domain 1098 30.3.4.3 Collagen Triple Helix Formation can be Nucleated at either End 1098 30.3.4.4 Hysteresis of Triple Helix Formation 1099 30.3.5 Influence of cis—trans Isomerase and Chaperones 1100 30.3.6 Mutations in Collagen Triple Helices Affect Proper Folding 1101 References 1101 31 Unfolding Induced by Mechanical Force Jane Clarke and Phil M. Williams 1111 31.1 Introduction 1111 31.2 Experimental Basics 1112 31.2.1 Instrumentation 1112 31.2.2 Sample Preparation 1113 31.2.3 Collecting Data 1114 31.2.4 Anatomy of a Force Trace 1115 31.2.5 Detecting Intermediates in a Force Trace 1115 31.2.6 Analyzing the Force Trace 1116 31.3 Analysis of Force Data 1117 31.3.1 Basic Theory behind Dynamic Force Spectroscopy 1117 31.3.2 The Ramp of Force Experiment 1119 31.3.3 The Golden Equation of DFS 1121 31.3.4 Nonlinear Loading 1122 31.3.4.1 The Worm-line Chain (WLC) 1123 31.3.5 Experiments under Constant Force 1124 31.3.6 Effect of Tandem Repeats on Kinetics 1125 31.3.7 Determining the Modal Force 1126 31.3.8 Comparing Behavior 1127 31.3.9 Fitting the Data 1127 31.4 Use of Complementary Techniques 1129 31.4.1 Protein Engineering 1130 31.4.1.1 Choosing Mutants 1130 31.4.1.2 Determining GD-N 1131 31.4.1.3 Determining GTS-N 1131 31.4.1.4 Interpreting the -values 1132 31.4.2 Computer Simulation 1133 31.5 Titin I27: A Case Study 1134 31.5.1 The Protein System 1134 31.5.2 The Unfolding Intermediate 1135 31.5.3 The Transition State 1136 31.5.4 The Relationship Between the Native and Transition States 1137 31.5.5 The Energy Landscape under Force 1139 31.6 Conclusions -- the Future 1139 References 1139 32 Molecular Dynamics Simulations to Study Protein Folding and Unfolding Amedeo Caflisch and Emanuele Paci 1143 32.1 Introduction 1143 32.2 Molecular Dynamics Simulations of Peptides and Proteins 1144 32.2.1 Folding of Structured Peptides 1144 32.2.1.1 Reversible Folding and Free Energy Surfaces 1144 32.2.1.2 Non-Arrhenius Temperature Dependence of the Folding Rate 1147 32.2.1.3 Denatured State and Levinthal Paradox 1148 32.2.1.4 Folding Events of Trp-cage 1149 32.2.2 Unfolding Simulations of Proteins 1150 32.2.2.1 High-temperature Simulations 1150 32.2.2.2 Biased Unfolding 1150 32.2.2.3 Forced Unfolding 1151 32.2.3 Determination of the Transition State Ensemble 1153 32.3 MD Techniques and Protocols 1155 32.3.1 Techniques to Improve Sampling 1155 32.3.1.1 Replica Exchange Molecular Dynamics 1155 32.3.1.2 Methods Based on Path Sampling 1157 32.3.2 MD with Restraints 1157 32.3.3 Distributed Computing Approach 1158 32.3.4 Implicit Solvent Models versus Explicit Water 1160 32.4 Conclusion 1162 References 1162 33 Molecular Dynamics Simulations of Proteins and Peptides: Problems, Achievements, and Perspectives Paul Tavan, Heiko Carstens, and Gerald Mathias 1170 33.1 Introduction 1170 33.2 Basic Physics of Protein Structure and Dynamics 1171 33.2.1 Protein Electrostatics 1172 33.2.2 Relaxation Times and Spatial Scales 1172 33.2.3 Solvent Environment 1173 33.2.4 Water 1174 33.2.5 Polarizability of the Peptide Groups and of Other Protein Components 1175 33.3 State of the Art 1177 33.3.1 Control of Thermodynamic Conditions 1177 33.3.2 Long-range Electrostatics 1177 33.3.3 Polarizability 1179 33.3.4 Higher Multipole Moments of the Molecular Components 1180 33.3.5 MM Models of Water 1181 33.3.6 Complexity of Protein--Solvent Systems and Consequences for MM-MD 1182 33.3.7 What about Successes of MD Methods? 1182 33.3.8 Accessible Time Scales and Accuracy Issues 1184 33.3.9 Continuum Solvent Models 1185 33.3.10 Are there Further Problems beyond Electrostatics and Structure Prediction? 1187 33.4 Conformational Dynamics of a Light-switchable Model Peptide 1187 33.4.1 Computational Methods 1188 33.4.2 Results and Discussion 1190 Summary 1194 Acknowledgments 1194 References 1194 Part II, Volume 1   Contributors of Part II LVIII 1 Paradigm Changes from ``Unboiling an Egg'' to ``Synthesizing a Rabbit'' Rainer Jaenicke 3 1.1 Protein Structure, Stability, and Self-organization 3 1.2 Autonomous and Assisted Folding and Association 6 1.3 Native, Intermediate, and Denatured States 11 1.4 Folding and Merging of Domains -- Association of Subunits 13 1.5 Limits of Reconstitution 19 1.6 In Vitro Denaturation-Renaturation vs. Folding in Vivo 21 1.7 Perspectives 24 Acknowledgements 26 References 26 2 Folding and Association of Multi-domain and Oligomeric Proteins Hauke Lilie and Robert Seckler 32 2.1 Introduction 32 2.2 Folding of Multi-domain Proteins 33 2.2.1 Domain Architecture 33 2.2.2 -Crystallin as a Model for a Two-domain Protein 35 2.2.3 The Giant Protein Titin 39 2.3 Folding and Association of Oligomeric Proteins 41 2.3.1 Why Oligomers? 41 2.3.2 Inter-subunit Interfaces 42 2.3.3 Domain Swapping 44 2.3.4 Stability of Oligomeric Proteins 45 2.3.5 Methods Probing Folding/Association 47 2.3.5.1 Chemical Cross-linking 47 2.3.5.2 Analytical Gel Filtration Chromatography 47 2.3.5.3 Scattering Methods 48 2.3.5.4 Fluorescence Resonance Energy Transfer 48 2.3.5.5 Hybrid Formation 48 2.3.6 Kinetics of Folding and Association 49 2.3.6.1 General Considerations 49 2.3.6.2 Reconstitution Intermediates 50 2.3.6.3 Rates of Association 52 2.3.6.4 Homo- Versus Heterodimerization 52 2.4 Renaturation versus Aggregation 54 2.5 Case Studies on Protein Folding and Association 54 2.5.1 Antibody Fragments 54 2.5.2 Trimeric Tail Spike Protein of Bacteriophage P22 59 2.6 Experimental Protocols 62 References 65 3 Studying Protein Folding in Vivo I. Marije Liscaljet, Bertrand Kleizen, and Ineke Braakman 73 3.1 Introduction 73 3.2 General Features in Folding Proteins Amenable to in Vivo Study 73 3.2.1 Increasing Compactness 76 3.2.2 Decreasing Accessibility to Different Reagents 76 3.2.3 Changes in Conformation 77 3.2.4 Assistance During Folding 78 3.3 Location-specific Features in Protein Folding 79 3.3.1 Translocation and Signal Peptide Cleavage 79 3.3.2 Glycosylation 80 3.3.3 Disulfide Bond Formation in the ER 81 3.3.4 Degradation 82 3.3.5 Transport from ER to Golgi and Plasma Membrane 83 3.4 How to Manipulate Protein Folding 84 3.4.1 Pharmacological Intervention (Low-molecular-weight Reagents) 84 3.4.1.1 Reducing and Oxidizing Agents 84 3.4.1.2 Calcium Depletion 84 3.4.1.3 ATP Depletion 85 3.4.1.4 Cross-linking 85 3.4.1.5 Glycosylation Inhibitors 85 3.4.2 Genetic Modifications (High-molecular-weight Manipulations) 86 3.4.2.1 Substrate Protein Mutants 86 3.4.2.2 Changing the Concentration or Activity of Folding Enzymes and Chaperones 87 3.5 Experimental Protocols 88 3.5.1 Protein-labeling Protocols 88 3.5.1.1 Basic Protocol Pulse Chase: Adherent Cells 88 3.5.1.2 Pulse Chase in Suspension Cells 91 3.5.2 (Co)-immunoprecipitation and Accessory Protocols 93 3.5.2.1 Immunoprecipitation 93 3.5.2.2 Co-precipitation with Calnexin ([84]; adapted from Ou et al. [85]) 94 3.5.2.3 Co-immunoprecipitation with Other Chaperones 95 3.5.2.4 Protease Resistance 95 3.5.2.5 Endo H Resistance 96 3.5.2.6 Cell Surface Expression Tested by Protease 96 3.5.3 SDS-PAGE [13] 97 Acknowledgements 98 References 98 4 Characterization of ATPase Cycles of Molecular Chaperones by Fluorescence and Transient Kinetic Methods Sandra Schlee and Jochen Reinstein 105 4.1 Introduction 105 4.1.1 Characterization of ATPase Cycles of Energy-transducing Systems 105 4.1.2 The Use of Fluorescent Nucleotide Analogues 106 4.1.2.1 Fluorescent Modifications of Nucleotides 106 4.1.2.2 How to Find a Suitable Analogue for a Specific Protein 108 4.2 Characterization of ATPase Cycles of Molecular Chaperones 109 4.2.1 Biased View 109 4.2.2 The ATPase Cycle of DnaK 109 4.2.3 The ATPase Cycle of the Chaperone Hsp90 109 4.2.4 The ATPase Cycle of the Chaperone ClpB 111 4.2.4.1 ClpB, an Oligomeric ATPase With Two AAA Modules Per Protomer 111 4.2.4.2 Nucleotide-binding Properties of NBD1 and NBD2 111 4.2.4.3 Cooperativity of ATP Hydrolysis and Interdomain Communication 114 4.3 Experimental Protocols 116 4.3.1 Synthesis of Fluorescent Nucleotide Analogues 116 4.3.1.1 Synthesis and Characterization of (P)MABA-ADP and (P)MABA-ATP 116 4.3.1.2 Synthesis and Characterization of N8-MABA Nucleotides 119 4.3.1.3 Synthesis of MANT Nucleotides 120 4.3.2 Preparation of Nucleotides and Proteins 121 4.3.2.1 Assessment of Quality of Nucleotide Stock Solution 121 4.3.2.2 Determination of the Nucleotide Content of Proteins 122 4.3.2.3 Nucleotide Depletion Methods 123 4.3.3 Steady-state ATPase Assays 124 4.3.3.1 Coupled Enzymatic Assay 124 4.3.3.2 Assays Based on [-32P]-ATP and TLC 125 4.3.3.3 Assays Based on Released Pi 125 4.3.4 Single-turnover ATPase Assays 126 4.3.4.1 Manual Mixing Procedures 126 4.3.4.2 Quenched Flow 127 4.3.5 Nucleotide-binding Measurements 127 4.3.5.1 Isothermal Titration Calorimetry 127 4.3.5.2 Equilibrium Dialysis 129 4.3.5.3 Filter Binding 129 4.3.5.4 Equilibrium Fluorescence Titration 130 4.3.5.5 Competition Experiments 132 4.3.6 Analytical Solutions of Equilibrium Systems 133 4.3.6.1 Quadratic Equation 133 4.3.6.2 Cubic Equation 134 4.3.6.3 Iterative Solutions 138 4.3.7 Time-resolved Binding Measurements 141 4.3.7.1 Introduction 141 4.3.7.2 One-step Irreversible Process 142 4.3.7.3 One-step Reversible Process 143 4.3.7.4 Reversible Second Order Reduced to Pseudo-first Order 144 4.3.7.5 Two Simultaneous Irreversible Pathways -- Partitioning 146 4.3.7.6 Two-step Consecutive (Sequential) Reaction 148 4.3.7.7 Two-step Binding Reactions 150 References 152 5 Analysis of Chaperone Function in Vitro Johannes Buchner and Stefan Walter 162 5.1 Introduction 162 5.2 Basic Functional Principles of Molecular Chaperones 164 5.2.1 Recognition of Nonnative Proteins 166 5.2.2 Induction of Conformational Changes in the Substrate 167 5.2.3 Energy Consumption and Regulation of Chaperone Function 169 5.3 Limits and Extensions of the Chaperone Concept 170 5.3.1 Co-chaperones 171 5.3.2 Specific Chaperones 171 5.4 Working with Molecular Chaperones 172 5.4.1 Natural versus Artificial Substrate Proteins 172 5.4.2 Stability of Chaperones 172 5.5 Assays to Assess and Characterize Chaperone Function 174 5.5.1 Generating Nonnative Conformations of Proteins 174 5.5.2 Aggregation Assays 174 5.5.3 Detection of Complexes Between Chaperone and Substrate 175 5.5.4 Refolding of Denatured Substrates 175 5.5.5 ATPase Activity and Effect of Substrate and Cofactors 176 5.6 Experimental Protocols 176 5.6.1 General Considerations 176 5.6.1.1 Analysis of Chaperone Stability 176 5.6.1.2 Generation of Nonnative Proteins 177 5.6.1.3 Model Substrates for Chaperone Assays 177 5.6.2 Suppression of Aggregation 179 5.6.3 Complex Formation between Chaperones and Polypeptide Substrates 183 5.6.4 Identification of Chaperone-binding Sites 184 5.6.5 Chaperone-mediated Refolding of Test Proteins 186 5.6.6 ATPase Activity 188 Acknowledgments 188 References 189 6 Physical Methods for Studies of Fiber Formation and Structure Thomas Scheibel and Louise Serpell 197 6.1 Introduction 197 6.2 Overview: Protein Fibers Formed in Vivo 198 6.2.1 Amyloid Fibers 198 6.2.2 Silks 199 6.2.3 Collagens 199 6.2.4 Actin, Myosin, and Tropomyosin Filaments 200 6.2.5 Intermediate Filaments/Nuclear Lamina 202 6.2.6 Fibrinogen/Fibrin 203 6.2.7 Microtubules 203 6.2.8 Elastic Fibers 204 6.2.9 Flagella and Pili 204 6.2.10 Filamentary Structures in Rod-like Viruses 205 6.2.11 Protein Fibers Used by Viruses and Bacteriophages to Bind to Their Hosts 206 6.3 Overview: Fiber Structures 206 6.3.1 Study of the Structure of -sheet-containing Proteins 207 6.3.1.1 Amyloid 207 6.3.1.2 Paired Helical Filaments 207 6.3.1.3 -Silks 207 6.3.1.4 -Sheet-containing Viral Fibers 208 6.3.2 -Helix-containing Protein Fibers 209 6.3.2.1 Collagen 209 6.3.2.2 Tropomyosin 210 6.3.2.3 Intermediate Filaments 210 6.3.3 Protein Polymers Consisting of a Mixture of Secondary Structure 211 6.3.3.1 Tubulin 211 6.3.3.2 Actin and Myosin Filaments 212 6.4 Methods to Study Fiber Assembly 213 6.4.1 Circular Dichroism Measurements for Monitoring Structural Changes Upon Fiber Assembly 213 6.4.1.1 Theory of CD 213 6.4.1.2 Experimental Guide to Measure CD Spectra and Structural Transition Kinetics 214 6.4.2 Intrinsic Fluorescence Measurements to Analyze Structural Changes 215 6.4.2.1 Theory of Protein Fluorescence 215 6.4.2.2 Experimental Guide to Measure Trp Fluorescence 216 6.4.3 Covalent Fluorescent Labeling to Determine Structural Changes of Proteins with Environmentally Sensitive Fluorophores 217 6.4.3.1 Theory on Environmental Sensitivity of Fluorophores 217 6.4.3.2 Experimental Guide to Labeling Proteins With Fluorophores 218 6.4.4 1-Anilino-8-Naphthalensulfonate (ANS) Binding to Investigate Fiber Assembly 219 6.4.4.1 Theory on Using ANS Fluorescence for Detecting Conformational Changes in Proteins 219 6.4.4.2 Experimental Guide to Using ANS for Monitoring Protein Fiber Assembly 220 6.4.5 Light Scattering to Monitor Particle Growth 220 6.4.5.1 Theory of Classical Light Scattering 221 6.4.5.2 Theory of Dynamic Light Scattering 221 6.4.5.3 Experimental Guide to Analyzing Fiber Assembly Using DLS 222 6.4.6 Field-flow Fractionation to Monitor Particle Growth 222 6.4.6.1 Theory of FFF 222 6.4.6.2 Experimental Guide to Using FFF for Monitoring Fiber Assembly 223 6.4.7 Fiber Growth-rate Analysis Using Surface Plasmon Resonance 223 6.4.7.1 Theory of SPR 223 6.4.7.2 Experimental Guide to Using SPR for Fiber-growth Analysis 224 6.4.8 Single-fiber Growth Imaging Using Atomic Force Microscopy 225 6.4.8.1 Theory of Atomic Force Microscopy 225 6.4.8.2 Experimental Guide for Using AFM to Investigate Fiber Growth 225 6.4.9 Dyes Specific for Detecting Amyloid Fibers 226 6.4.9.1 Theory on Congo Red and Thioflavin T Binding to Amyloid 226 6.4.9.2 Experimental Guide to Detecting Amyloid Fibers with CR and Thioflavin Binding 227 6.5 Methods to Study Fiber Morphology and Structure 228 6.5.1 Scanning Electron Microscopy for Examining the Low-resolution Morphology of a Fiber Specimen 228 6.5.1.1 Theory of SEM 228 6.5.1.2 Experimental Guide to Examining Fibers by SEM 229 6.5.2 Transmission Electron Microscopy for Examining Fiber Morphology and Structure 230 6.5.2.1 Theory of TEM 230 6.5.2.2 Experimental Guide to Examining Fiber Samples by TEM 231 6.5.3 Cryo-electron Microscopy for Examination of the Structure of Fibrous Proteins 232 6.5.3.1 Theory of Cryo-electron Microscopy 232 6.5.3.2 Experimental Guide to Preparing Proteins for Cryo-electron Microscopy 233 6.5.3.3 Structural Analysis from Electron Micrographs 233 6.5.4 Atomic Force Microscopy for Examining the Structure and Morphology of Fibrous Proteins 234 6.5.4.1 Experimental Guide for Using AFM to Monitor Fiber Morphology 234 6.5.5 Use of X-ray Diffraction for Examining the Structure of Fibrous Proteins 236 6.5.5.1 Theory of X-Ray Fiber Diffraction 236 6.5.5.2 Experimental Guide to X-Ray Fiber Diffraction 237 6.5.6 Fourier Transformed Infrared Spectroscopy 239 6.5.6.1 Theory of FTIR 239 6.5.6.2 Experimental Guide to Determining Protein Conformation by FTIR 240 6.6 Concluding Remarks 241 Acknowledgements 242 References 242 7 Protein Unfolding in the Cell Prakash Koodathingal, Neil E. Jaffe, and Andreas Matouschek 254 7.1 Introduction 254 7.2 Protein Translocation Across Membranes 254 7.2.1 Compartmentalization and Unfolding 254 7.2.2 Mitochondria Actively Unfold Precursor Proteins 256 7.2.3 The Protein Import Machinery of Mitochondria 257 7.2.4 Specificity of Unfolding 259 7.2.5 Protein Import into Other Cellular Compartments 259 7.3 Protein Unfolding and Degradation by ATP-dependent Proteases 260 7.3.1 Structural Considerations of Unfoldases Associated With Degradation 260 7.3.2 Unfolding Is Required for Degradation by ATP-dependent Proteases 261 7.3.3 The Role of ATP and Models of Protein Unfolding 262 7.3.4 Proteins Are Unfolded Sequentially and Processively 263 7.3.5 The Influence of Substrate Structure on the Degradation Process 264 7.3.6 Unfolding by Pulling 264 7.3.7 Specificity of Degradation 265 7.4 Conclusions 266 7.5 Experimental Protocols 266 7.5.1 Size of Import Channels in the Outer and Inner Membranes of Mitochondria 266 7.5.2 Structure of Precursor Proteins During Import into Mitochondria 266 7.5.3 Import of Barnase Mutants 267 7.5.4 Protein Degradation by ATP-dependent Proteases 267 7.5.5 Use of Multi-domain Substrates 268 7.5.6 Studies Using Circular Permutants 268 References 269 8 Natively Disordered Proteins Gary W. Daughdrill, Gary J. Pielak, Vladimir N. Uversky, Marc S. Cortese, and A. Keith Dunker 275 8.1 Introduction 275 8.1.1 The Protein Structure-Function Paradigm 275 8.1.2 Natively Disordered Proteins 277 8.1.3 A New Protein Structure-Function Paradigm 280 8.2 Methods Used to Characterize Natively Disordered Proteins 281 8.2.1 NMR Spectroscopy 281 8.2.1.1 Chemical Shifts Measure the Presence of Transient Secondary Structure 282 8.2.1.2 Pulsed Field Gradient Methods to Measure Translational Diffusion 284 8.2.1.3 NMR Relaxation and Protein Flexibility 284 8.2.1.4 Using the Model-free Analysis of Relaxation Data to Estimate Internal Mobility and Rotational Correlation Time 285 8.2.1.5 Using Reduced Spectral Density Mapping to Assess the Amplitude and Frequencies of Intramolecular Motion 286 8.2.1.6 Characterization of the Dynamic Structures of Natively Disordered Proteins Using NMR 287 8.2.2 X-ray Crystallography 288 8.2.3 Small Angle X-ray Diffraction and Hydrodynamic Measurements 293 8.2.4 Circular Dichroism Spectropolarimetry 297 8.2.5 Infrared and Raman Spectroscopy 299 8.2.6 Fluorescence Methods 301 8.2.6.1 Intrinsic Fluorescence of Proteins 301 8.2.6.2 Dynamic Quenching of Fluorescence 302 8.2.6.3 Fluorescence Polarization and Anisotropy 303 8.2.6.4 Fluorescence Resonance Energy Transfer 303 8.2.6.5 ANS Fluorescence 305 8.2.7 Conformational Stability 308 8.2.7.1 Effect of Temperature on Proteins with Extended Disorder 309 8.2.7.2 Effect of pH on Proteins with Extended Disorder 309 8.2.8 Mass Spectrometry-based High-resolution Hydrogen-Deuterium Exchange 309 8.2.9 Protease Sensitivity 311 8.2.10 Prediction from Sequence 313 8.2.11 Advantage of Multiple Methods 314 8.3 Do Natively Disordered Proteins Exist Inside Cells? 315 8.3.1 Evolution of Ordered and Disordered Proteins Is Fundamentally Different 315 8.3.1.1 The Evolution of Natively Disordered Proteins 315 8.3.1.2 Adaptive Evolution and Protein Flexibility 317 8.3.1.3 Phylogeny Reconstruction and Protein Structure 318 8.3.2 Direct Measurement by NMR 320 8.4 Functional Repertoire 322 8.4.1 Molecular Recognition 322 8.4.1.1 The Coupling of Folding and Binding 322 8.4.1.2 Structural Plasticity for the Purpose of Functional Plasticity 323 8.4.1.3 Systems Where Disorder Increases Upon Binding 323 8.4.2 Assembly/Disassembly 325 8.4.3 Highly Entropic Chains 325 8.4.4 Protein Modification 327 8.5 Importance of Disorder for Protein Folding 328 8.6 Experimental Protocols 331 8.6.1 NMR Spectroscopy 331 8.6.1.1 General Requirements 331 8.6.1.2 Measuring Transient Secondary Structure in Secondary Chemical Shifts 332 8.6.1.3 Measuring the Translational Diffusion Coefficient Using Pulsed Field Gradient Diffusion Experiments 332 8.6.1.4 Relaxation Experiments 332 8.6.1.5 Relaxation Data Analysis Using Reduced Spectral Density Mapping 333 8.6.1.6 In-cell NMR 334 8.6.2 X-ray Crystallography 334 8.6.3 Circular Dichroism Spectropolarimetry 336 Acknowledgements 337 References 337 9 The Catalysis of Disulfide Bond Formation in Prokaryotes Jean-Francois Collet and James C. Bardwell 358 9.1 Introduction 358 9.2 Disulfide Bond Formation in the E. coli Periplasm 358 9.2.1 A Small Bond, a Big Effect 358 9.2.2 Disulfide Bond Formation Is a Catalyzed Process 359 9.2.3 DsbA, a Protein-folding Catalyst 359 9.2.4 How is DsbA Re-oxidized? 361 9.2.5 From Where Does the Oxidative Power of DsbB Originate? 361 9.2.6 How Are Disulfide Bonds Transferred From DsbB to DsbA? 362 9.2.7 How Can DsbB Generate Disulfide by Quinone Reduction? 364 9.3 Disulfide Bond Isomerization 365 9.3.1 The Protein Disulfide Isomerases DsbC and DsbG 365 9.3.2 Dimerization of DsbC and DsbG Is Important for Isomerase and Chaperone Activity 366 9.3.3 Dimerization Protects from DsbB Oxidation 367 9.3.4 Import of Electrons from the Cytoplasm: DsbD 367 9.3.5 Conclusions 369 9.4 Experimental Protocols 369 9.4.1 Oxidation-reduction of a Protein Sample 369 9.4.2 Determination of the Free Thiol Content of a Protein 370 9.4.3 Separation by HPLC 371 9.4.4 Tryptophan Fluorescence 372 9.4.5 Assay of Disulfide Oxidase Activity 372 References 373 10 Catalysis of Peptidyl-prolyl cis/trans Isomerization by Enzymes 377 Gunter Fischer 10.1 Introduction 377 10.2 Peptidyl-prolyl cis/trans Isomerization 379 10.3 Monitoring Peptidyl-prolyl cis/trans Isomerase Activity 383 10.4 Prototypical Peptidyl-prolyl cis/trans Isomerases 388 10.4.1 General Considerations 388 10.4.2 Prototypic Cyclophilins 390 10.4.3 Prototypic FK506-binding Proteins 394 10.4.4 Prototypic Parvulins 397 10.5 Concluding Remarks 399 10.6 Experimental Protocols 399 10.6.1 PPIase Assays: Materials 399 10.6.2 PPIase Assays: Equipment 400 10.6.3 Assaying Procedure: Protease-coupled Spectrophotometric Assay 400 10.6.4 Assaying Procedure: Protease-free Spectrophotometric Assay 401 References 401 11 Secondary Amide Peptide Bond cis/trans Isomerization in Polypeptide Backbone Restructuring: Implications for Catalysis Cordelia Schiene-Fischer and Christian Lücke 415 11.1 Introduction 415 11.2 Monitoring Secondary Amide Peptide Bond cis/trans Isomerization 416 11.3 Kinetics and Thermodynamics of Secondary Amide Peptide Bond cis/trans Isomerization 418 11.4 Principles of DnaK Catalysis 420 11.5 Concluding Remarks 423 11.6 Experimental Protocols 424 11.6.1 Stopped-flow Measurements of Peptide Bond cis/trans Isomerization 424 11.6.2 Two-dimensional 1H-NMR Exchange Experiments 425 References 426 12 Ribosome-associated Proteins Acting on Newly Synthesized Polypeptide Chains Sabine Rospert, Matthias Gautschi, Magdalena Rakwalska, and Uta Raue 429 12.1 Introduction 429 12.2 Signal Recognition Particle, Nascent Polypeptide--associated Complex, and Trigger Factor 432 12.2.1 Signal Recognition Particle 432 12.2.2 An Interplay between Eukaryotic SRP and Nascent Polypeptide--associated Complex? 435 12.2.3 Interplay between Bacterial SRP and Trigger Factor? 435 12.2.4 Functional Redundancy: TF and the Bacterial Hsp70 Homologue DnaK 436 12.3 Chaperones Bound to the Eukaryotic Ribosome: Hsp70 and Hsp40 Systems 436 12.3.1 Sis1p and Ssa1p: an Hsp70/Hsp40 System Involved in Translation Initiation? 437 12.3.2 Ssb1/2p, an Hsp70 Homologue Distributed Between Ribosomes and Cytosol 438 12.3.3 Function of Ssb1/2p in Degradation and Protein Folding 439 12.3.4 Zuotin and Ssz1p: a Stable Chaperone Complex Bound to the Yeast Ribosome 440 12.3.5 A Functional Chaperone Triad Consisting of Ssb1/2p, Ssz1p, and Zuotin 440 12.3.6 Effects of Ribosome-bound Chaperones on the Yeast Prion [PSI+] 442 12.4 Enzymes Acting on Nascent Polypeptide Chains 443 12.4.1 Methionine Aminopeptidases 443 12.4.2 N-acetyltransferases 444 12.5 A Complex Arrangement at the Yeast Ribosomal Tunnel Exit 445 12.6 Experimental Protocols 446 12.6.1 Purification of Ribosome-associated Protein Complexes from Yeast 446 12.6.2 Growth of Yeast and Preparation of Ribosome-associated Proteins by High-salt Treatment of Ribosomes 447 12.6.3 Purification of NAC and RAC 448 References 449 Part II, Volume 2   13 The Role of Trigger Factor in Folding of Newly Synthesized Proteins Elke Deuerling, Thomas Rauch, Holger Patzelt, and Bernd Bukau 459 13.1 Introduction 459 13.2 In Vivo Function of Trigger Factor 459 13.2.1 Discovery 459 13.2.2 Trigger Factor Cooperates With the DnaK Chaperone in the Folding of Newly Synthesized Cytosolic Proteins 460 13.2.3 In Vivo Substrates of Trigger Factor and DnaK 461 13.2.4 Substrate Specificity of Trigger Factor 463 13.3 Structure--Function Analysis of Trigger Factor 465 13.3.1 Domain Structure and Conservation 465 13.3.2 Quaternary Structure 468 13.3.3 PPIase and Chaperone Activity of Trigger Factor 469 13.3.4 Importance of Ribosome Association 470 13.4 Models of the Trigger Factor Mechanism 471 13.5 Experimental Protocols 473 13.5.1 Trigger Factor Purification 473 13.5.2 GAPDH Trigger Factor Activity Assay 475 13.5.3 Modular Cell-free E. coli Transcription/Translation System 475 13.5.4 Isolation of Ribosomes and Add-back Experiments 483 13.5.5 Cross-linking Techniques 485 References 485 14 Cellular Functions of Hsp70 Chaperones Elizabeth A. Craig and Peggy Huang 490 14.1 Introduction 490 14.2 ``Soluble'' Hsp70s/J-proteins Function in General Protein Folding 492 14.2.1 The Soluble Hsp70 of E. coli, DnaK 492 14.2.2 Soluble Hsp70s of Major Eukaryotic Cellular Compartments 493 14.2.2.1 Eukaryotic Cytosol 493 14.2.2.2 Matrix of Mitochondria 494 14.2.2.3 Lumen of the Endoplasmic Reticulum 494 14.3 ``Tethered'' Hsp70s/J-proteins: Roles in Protein Folding on the Ribosome and in Protein Translocation 495 14.3.1 Membrane-tethered Hsp70/J-protein 495 14.3.2 Ribosome-associated Hsp70/J-proteins 496 14.4 Modulating of Protein Conformation by Hsp70s/J-proteins 498 14.4.1 Assembly of Fe/S Centers 499 14.4.2 Uncoating of Clathrin-coated Vesicles 500 14.4.3 Regulation of the Heat Shock Response 501 14.4.4 Regulation of Activity of DNA Replication-initiator Proteins 502 14.5 Cases of a Single Hsp70 Functioning With Multiple J-Proteins 504 14.6 Hsp70s/J-proteins -- When an Hsp70 Maybe Isn't Really a Chaperone 504 14.6.1 The Ribosome-associated ``Hsp70'' Ssz1 505 14.6.2 Mitochondrial Hsp70 as the Regulatory Subunit of an Endonuclease 506 14.7 Emerging Concepts and Unanswered Questions 507 References 507 15 Regulation of Hsp70 Chaperones by Co-chaperones Matthias P. Mayer and Bernd Bukau 516 15.1 Introduction 516 15.2 Hsp70 Proteins 517 15.2.1 Structure and Conservation 517 15.2.2 ATPase Cycle 519 15.2.3 Structural Investigations 521 15.2.4 Interactions With Substrates 522 15.3 J-domain Protein Family 526 15.3.1 Structure and Conservation 526 15.3.2 Interaction With Hsp70s 530 15.3.3 Interactions with Substrates 532 15.4 Nucleotide Exchange Factors 534 15.4.1 GrpE: Structure and Interaction with DnaK 534 15.4.2 Nucleotide Exchange Reaction 535 15.4.3 Bag Family: Structure and Interaction With Hsp70 536 15.4.4 Relevance of Regulated Nucleotide Exchange for Hsp70s 538 15.5 TPR Motifs Containing Co-chaperones of Hsp70 540 15.5.1 Hip 541 15.5.2 Hop 542 15.5.3 Chip 543 15.6 Concluding Remarks 544 15.7 Experimental Protocols 544 15.7.1 Hsp70s 544 15.7.2 J-Domain Proteins 545 15.7.3 GrpE 546 15.7.4 Bag-1 547 15.7.5 Hip 548 15.7.6 Hop 549 15.7.7 Chip 549 References 550 16 Protein Folding in the Endoplasmic Reticulum Via the Hsp70 Family Ying Shen, Kyung Tae Chung, and Linda M. Hendershot 563 16.1 Introduction 563 16.2 BiP Interactions with Unfolded Proteins 564 16.3 ER-localized DnaJ Homologues 567 16.4 ER-localized Nucleotide-exchange/releasing Factors 571 16.5 Organization and Relative Levels of Chaperones in the ER 572 16.6 Regulation of ER Chaperone Levels 573 16.7 Disposal of BiP-associated Proteins That Fail to Fold or Assemble 575 16.8 Other Roles of BiP in the ER 576 16.9 Concluding Comments 576 16.10 Experimental Protocols 577 16.10.1 Production of Recombinant ER Proteins 577 16.10.1.1 General Concerns 577 16.10.1.2 Bacterial Expression 578 16.10.1.3 Yeast Expression 580 16.10.1.4 Baculovirus 581 16.10.1.5 Mammalian Cells 583 16.10.2 Yeast Two-hybrid Screen for Identifying Interacting Partners of ER Proteins 586 16.10.3 Methods for Determining Subcellular Localization, Topology, and Orientation of Proteins 588 16.10.3.1 Sequence Predictions 588 16.10.3.2 Immunofluorescence Staining 589 16.10.3.3 Subcellular Fractionation 589 16.10.3.4 Determination of Topology 590 16.10.3.5 N-linked Glycosylation 592 16.10.4 Nucleotide Binding, Hydrolysis, and Exchange Assays 594 16.10.4.1 Nucleotide-binding Assays 594 16.10.4.2 ATP Hydrolysis Assays 596 16.10.4.3 Nucleotide Exchange Assays 597 16.10.5 Assays for Protein--Protein Interactions in Vitro/in Vivo 599 16.10.5.1 In Vitro GST Pull-down Assay 599 16.10.5.2 Co-immunoprecipitation 600 16.10.5.3 Chemical Cross-linking 600 16.10.5.4 Yeast Two-hybrid System 601 16.10.6 In Vivo Folding, Assembly, and Chaperone-binding Assays 601 16.10.6.1 Monitoring Oxidation of Intrachain Disulfide Bonds 601 16.10.6.2 Detection of Chaperone Binding 602 Acknowledgements 603 References 603 17 Quality Control In Glycoprotein Folding E. Sergio Trombetta and Armando J. Parodi 617 17.1 Introduction 617 17.2 ER N-glycan Processing Reactions 617 17.3 The UDP-Glc:Glycoprotein Glucosyltransferase 619 17.4 Protein Folding in the ER 621 17.5 Unconventional Chaperones (Lectins) Are Present in the ER Lumen 621 17.6 In Vivo Glycoprotein-CNX/CRT Interaction 623 17.7 Effect of CNX/CRT Binding on Glycoprotein Folding and ER Retention 624 17.8 Glycoprotein-CNX/CRT Interaction Is Not Essential for Unicellular Organisms and Cells in Culture 627 17.9 Diversion of Misfolded Glycoproteins to Proteasomal Degradation 629 17.10 Unfolding Irreparably Misfolded Glycoproteins to Facilitate Proteasomal Degradation 632 17.11 Summary and Future Directions 633 17.12 Characterization of N-glycans from Glycoproteins 634 17.12.1 Characterization of N-glycans Present in Immunoprecipitated Samples 634 17.12.2 Analysis of Radio-labeled N-glycans 636 17.12.3 Extraction and Analysis of Protein-bound N-glycans 636 17.12.4 GII and GT Assays 637 17.12.4.1 Assay for GII 637 17.12.4.2 Assay for GT 638 17.12.5 Purification of GII and GT from Rat Liver 639 References 641 18 Procollagen Biosynthesis in Mammalian Cells Mohammed Tasab and Neil J. Bulleid 649 18.1 Introduction 649 18.1.1 Variety and Complexity of Collagen Proteins 649 18.1.2 Fibrillar Procollagen 650 18.1.3 Expression of Fibrillar Collagens 650 18.2 The Procollagen Biosynthetic Process: An Overview 651 18.3 Disulfide Bonding in Procollagen Assembly 653 18.4 The Influence of Primary Amino Acid Sequence on Intracellular Procollagen Folding 654 18.4.1 Chain Recognition and Type-specific Assembly 654 18.4.2 Assembly of Multi-subunit Proteins 654 18.4.3 Coordination of Type-specific Procollagen Assembly and Chain Selection 655 18.4.4 Hypervariable Motifs: Components of a Recognition Mechanism That Distinguishes Between Procollagen Chains? 656 18.4.5 Modeling the C-propeptide 657 18.4.6 Chain Association 657 18.5 Posttranslational Modifications That Affect Procollagen Folding 658 18.5.1 Hydroxylation and Triple-helix Stability 658 18.6 Procollagen Chaperones 658 18.6.1 Prolyl 4-Hydroxylase 658 18.6.2 Protein Disulfide Isomerase 659 18.6.3 Hsp47 660 18.6.4 PPI and BiP 661 18.7 Analysis of Procollagen Folding 662 18.8 Experimental Part 663 18.8.1 Materials Required 663 18.8.2 Experimental Protocols 664 References 668 19 Redox Regulation of Chaperones Jörg H. Hoffmann and Ursula Jakob 677 19.1 Introduction 677 19.2 Disulfide Bonds as Redox-Switches 677 19.2.1 Functionality of Disulfide Bonds 677 19.2.2 Regulatory Disulfide Bonds as Functional Switches 679 19.2.3 Redox Regulation of Chaperone Activity 680 19.3 Prokaryotic Hsp33: A Chaperone Activated by Oxidation 680 19.3.1 Identification of a Redox-regulated Chaperone 680 19.3.2 Activation Mechanism of Hsp33 681 19.3.3 The Crystal Structure of Active Hsp33 682 19.3.4 The Active Hsp33-Dimer: An Efficient Chaperone Holdase 683 19.3.5 Hsp33 is Part of a Sophisticated Multi-chaperone Network 684 19.4 Eukaryotic Protein Disulfide Isomerase (PDI): Redox Shuffling in the ER 685 19.4.1 PDI, A Multifunctional Enzyme in Eukaryotes 685 19.4.2 PDI and Redox Regulation 687 19.5 Concluding Remarks and Outlook 688 19.6 Appendix -- Experimental Protocols 688 19.6.1 How to Work With Redox-regulated Chaperones in Vitro 689 19.6.1.1 Preparation of the Reduced Protein Species 689 19.6.1.2 Preparation of the Oxidized Protein Species 690 19.6.1.3 In Vitro Thiol Trapping to Monitor the Redox State of Proteins 691 19.6.2 Thiol Coordinating Zinc Centers as Redox Switches 691 19.6.2.1 PAR-PMPS Assay to Quantify Zinc 691 19.6.2.2 Determination of Zinc-binding Constants 692 19.6.3 Functional Analysis of Redox-regulated Chaperones in Vitro/in Vivo 693 19.6.3.1 Chaperone Activity Assays 693 19.6.3.2 Manipulating and Analyzing Redox Conditions in Vivo 694 Acknowledgements 694 References 694 20 The E. coli GroE Chaperone Steven G. Burston and Stefan Walter 699 20.1 Introduction 699 20.2 The Structure of GroEL 699 20.3 The Structure of GroEL-ATP 700 20.4 The Structure of GroES and its Interaction with GroEL 701 20.5 The Interaction Between GroEL and Substrate Polypeptides 702 20.6 GroEL is a Complex Allosteric Macromolecule 703 20.7 The Reaction Cycle of the GroE Chaperone 705 20.8 The Effect of GroE on Protein-folding Pathways 708 20.9 Future Perspectives 710 20.10 Experimental Protocols 710 Acknowledgments 719 References 719 21 Structure and Function of the Cytosolic Chaperonin CCT José M. Valpuesta, José L. Carrascosa, and Keith R. Willison 725 21.1 Introduction 725 21.2 Structure and Composition of CCT 726 21.3 Regulation of CCT Expression 729 21.4 Functional Cycle of CCT 730 21.5 Folding Mechanism of CCT 731 21.6 Substrates of CCT 735 21.7 Co-chaperones of CCT 739 21.8 Evolution of CCT 741 21.9 Concluding Remarks 743 21.10 Experimental Protocols 743 21.10.1 Purification 743 21.10.2 ATP Hydrolysis Measurements 744 21.10.3 CCT Substrate-binding and Folding Assays 744 21.10.4 Electron Microscopy and Image Processing 744 References 747 22 Structure and Function of GimC/Prefoldin Katja Siegers, Andreas Bracher, and Ulrich Hartl 756 22.1 Introduction 756 22.2 Evolutionary Distribution of GimC/Prefoldin 757 22.3 Structure of the Archaeal GimC/Prefoldin 757 22.4 Complexity of the Eukaryotic/Archaeal GimC/Prefoldin 759 22.5 Functional Cooperation of GimC/Prefoldin With the Eukaryotic Chaperonin TRiC/CCT 761 22.6 Experimental Protocols 764 22.6.1 Actin-folding Kinetics 764 22.6.2 Prevention of Aggregation (Light-scattering) Assay 765 22.6.3 Actin-binding Assay 765 Acknowledgements 766 References 766 23 Hsp90: From Dispensable Heat Shock Protein to Global Player Klaus Richter, Birgit Meinlschmidt, and Johannes Buchner 768 23.1 Introduction 768 23.2 The Hsp90 Family in Vivo 768 23.2.1 Evolutionary Relationships within the Hsp90 Gene Family 768 23.2.2 In Vivo Functions of Hsp90 769 23.2.3 Regulation of Hsp90 Expression and Posttranscriptional Activation 772 23.2.4 Chemical Inhibition of Hsp90 773 23.2.5 Identification of Natural Hsp90 Substrates 774 23.3 In Vitro Investigation of the Chaperone Hsp90 775 23.3.1 Hsp90: A Special Kind of ATPase 775 23.3.2 The ATPase Cycle of Hsp90 780 23.3.3 Interaction of Hsp90 with Model Substrate Proteins 781 23.3.4 Investigating Hsp90 Substrate Interactions Using Native Substrates 783 23.4 Partner Proteins: Does Complexity Lead to Specificity? 784 23.4.1 Hop, p23, and PPIases: The Chaperone Cycle of Hsp90 784 23.4.2 Hop/Sti1: Interactions Mediated by TPR Domains 787 23.4.3 p23/Sba1: Nucleotide-specific Interaction with Hsp90 789 23.4.4 Large PPIases: Conferring Specificity to Substrate Localization? 790 23.4.5 Pp5: Facilitating Dephosphorylation 791 23.4.6 Cdc37: Building Complexes with Kinases 792 23.4.7 Tom70: Chaperoning Mitochondrial Import 793 23.4.8 CHIP and Sgt1: Multiple Connections to Protein Degradation 793 23.4.9 Aha1 and Hch1: Just Stimulating the ATPase? 794 23.4.10 Cns1, Sgt2, and Xap2: Is a TPR Enough to Become an Hsp90 Partner? 796 23.5 Outlook 796 23.6 Appendix -- Experimental Protocols 797 23.6.1 Calculation of Phylogenetic Trees Based on Protein Sequences 797 23.6.2 Investigating the in Vivo Effect of Hsp90 Mutations in S. cerevisiae 797 23.6.3 Well-characterized Hsp90 Mutants 798 23.6.4 Investigating Activation of Heterologously Expressed Src Kinase in S. cerevisiae 800 23.6.5 Investigation of Heterologously Expressed Glucocorticoid Receptor in S. cerevisiae 800 23.6.6 Investigation of Chaperone Activity 801 23.6.7 Analysis of the ATPase Activity of Hsp90 802 23.6.8 Detecting Specific Influences on Hsp90 ATPase Activity 803 23.6.9 Investigation of the Quaternary Structure by SEC-HPLC 804 23.6.10 Investigation of Binding Events Using Changes of the Intrinsic Fluorescence 806 23.6.11 Investigation of Binding Events Using Isothermal Titration Calorimetry 807 23.6.12 Investigation of Protein-Protein Interactions Using Cross-linking 807 23.6.13 Investigation of Protein-Protein Interactions Using Surface Plasmon Resonance Spectroscopy 808 Acknowledgements 810 References 810 24 Small Heat Shock Proteins: Dynamic Players in the Folding Game Franz Narberhaus and Martin Haslbeck 830 24.1 Introduction 830 24.2 -Crystallins and the Small Heat Shock Protein Family: Diverse Yet Similar 830 24.3 Cellular Functions of -Hsps 831 24.3.1 Chaperone Activity in Vitro 831 24.3.2 Chaperone Function in Vivo 835 24.3.3 Other Functions 836 24.4 The Oligomeric Structure of -Hsps 837 24.5 Dynamic Structures as Key to Chaperone Activity 839 24.6 Experimental Protocols 840 24.6.1 Purification of sHsps 840 24.6.2 Chaperone Assays 843 24.6.3 Monitoring Dynamics of sHsps 846 Acknowledgements 847 References 848 25 Alpha-crystallin: Its Involvement in Suppression of Protein Aggregation and Protein Folding Joseph Horwitz 858 25.1 Introduction 858 25.2 Distribution of Alpha-crystallin in the Various Tissues 858 25.3 Structure 859 25.4 Phosphorylation and Other Posttranslation Modification 860 25.5 Binding of Target Proteins to Alpha-crystallin 861 25.6 The Function of Alpha-crystallin 863 25.7 Experimental Protocols 863 25.7.1 Preparation of Alpha-crystallin 863 Acknowledgements 870 References 870 26 Transmembrane Domains in Membrane Protein Folding, Oligomerization, and Function Anja Ridder and Dieter Langosch 876 26.1 Introduction 876 26.1.1 Structure of Transmembrane Domains 876 26.1.2 The Biosynthetic Route towards Folded and Oligomeric Integral Membrane Proteins 877 26.1.3 Structure and Stability of TMSs 878 26.1.3.1 Amino Acid Composition of TMSs and Flanking Regions 878 26.1.3.2 Stability of Transmembrane Helices 879 26.2 The Nature of Transmembrane Helix-Helix Interactions 880 26.2.1 General Considerations 880 26.2.1.1 Attractive Forces within Lipid Bilayers 880 26.2.1.2 Forces between Transmembrane Helices 881 26.2.1.3 Entropic Factors Influencing Transmembrane Helix--Helix Interactions 882 26.2.2 Lessons from Sequence Analyses and High-resolution Structures 883 26.2.3 Lessons from Bitopic Membrane Proteins 886 26.2.3.1 Transmembrane Segments Forming Right-handed Pairs 886 26.2.3.2 Transmembrane Segments Forming Left-handed Assemblies 889 26.2.4 Selection of Self-interacting TMSs from Combinatorial Libraries 892 26.2.5 Role of Lipids in Packing/Assembly of Membrane Proteins 893 26.3 Conformational Flexibility of Transmembrane Segments 895 26.4 Experimental Protocols 897 26.4.1 Biochemical and Biophysical Techniques 897 26.4.1.1 Visualization of Oligomeric States by Electrophoretic Techniques 898 26.4.1.2 Hydrodynamic Methods 899 26.4.1.3 Fluorescence Resonance Transfer 900 26.4.2 Genetic Assays 901 26.4.2.1 The ToxR System 901 26.4.2.2 Other Genetic Assays 902 26.4.3 Identification of TMS-TMS Interfaces by Mutational Analysis 903 References 904 Part II, Volume 3   27 SecB Arnold J. M. Driessen, Janny de Wit, and Nico Nouwen 919 27.1 Introduction 919 27.2 Selective Binding of Preproteins by SecB 920 27.3 SecA-SecB Interaction 925 27.4 Preprotein Transfer from SecB to SecA 928 27.5 Concluding Remarks 929 27.6 Experimental Protocols 930 27.6.1 How to Analyze SecB-Preprotein Interactions 930 27.6.2 How to Analyze SecB-SecA Interaction 931 Acknowledgements 932 References 933 28 Protein Folding in the Periplasm and Outer Membrane of E. coli Michael Ehrmann 938 28.1 Introduction 938 28.2 Individual Cellular Factors 940 28.2.1 The Proline Isomerases FkpA, PpiA, SurA, and PpiD 941 28.2.1.1 FkpA 942 28.2.1.2 PpiA 942 28.2.1.3 SurA 943 28.2.1.4 PpiD 943 28.2.2 Skp 944 28.2.3 Proteases and Protease/Chaperone Machines 945 28.2.3.1 The HtrA Family of Serine Proteases 946 28.2.3.2 E. coli HtrAs 946 28.2.3.3 DegP and DegQ 946 28.2.3.4 DegS 947 28.2.3.5 The Structure of HtrA 947 28.2.3.6 Other Proteases 948 28.3 Organization of Folding Factors into Pathways and Networks 950 28.3.1 Synthetic Lethality and Extragenic High-copy Suppressors 950 28.3.2 Reconstituted in Vitro Systems 951 28.4 Regulation 951 28.4.1 The Sigma E Pathway 951 28.4.2 The Cpx Pathway 952 28.4.3 The Bae Pathway 953 28.5 Future Perspectives 953 28.6 Experimental Protocols 954 28.6.1 Pulse Chase Immunoprecipitation 954 Acknowledgements 957 References 957 29 Formation of Adhesive Pili by the Chaperone-Usher Pathway Michael Vetsch and Rudi Glockshuber 965 29.1 Basic Properties of Bacterial, Adhesive Surface Organelles 965 29.2 Structure and Function of Pilus Chaperones 970 29.3 Structure and Folding of Pilus Subunits 971 29.4 Structure and Function of Pilus Ushers 973 29.5 Conclusions and Outlook 976 29.6 Experimental Protocols 977 29.6.1 Test for the Presence of Type 1 Piliated E. coli Cells 977 29.6.2 Functional Expression of Pilus Subunits in the E. coli Periplasm 977 29.6.3 Purification of Pilus Subunits from the E. coli Periplasm 978 29.6.4 Preparation of Ushers 979 Acknowledgements 979 References 980 30 Unfolding of Proteins During Import into Mitochondria Walter Neupert, Michael Brunner, and Kai Hell 987 30.1 Introduction 987 30.2 Translocation Machineries and Pathways of the Mitochondrial Protein Import System 988 30.2.1 Import of Proteins Destined for the Mitochondrial Matrix 990 30.3 Import into Mitochondria Requires Protein Unfolding 993 30.4 Mechanisms of Unfolding by the Mitochondrial Import Motor 995 30.4.1 Targeted Brownian Ratchet 995 30.4.2 Power-stroke Model 995 30.5 Studies to Discriminate between the Models 996 30.5.1 Studies on the Unfolding of Preproteins 996 30.5.1.1 Comparison of the Import of Folded and Unfolded Proteins 996 30.5.1.2 Import of Preproteins With Different Presequence Lengths 999 30.5.1.3 Import of Titin Domains 1000 30.5.1.4 Unfolding by the Mitochondrial Membrane Potential 1000 30.5.2 Mechanistic Studies of the Import Motor 1000 30.5.2.1 Brownian Movement of the Polypeptide Within the Import Channel 1000 30.5.2.2 Recruitment of mtHsp70 by Tim44 1001 30.5.2.3 Import Without Recruitment of mtHsp70 by Tim44 1002 30.5.2.4 MtHsp70 Function in the Import Motor 1003 30.6 Discussion and Perspectives 1004 30.7 Experimental Protocols 1006 30.7.1 Protein Import Into Mitochondria in Vitro 1006 30.7.2 Stabilization of the DHFR Domain by Methotrexate 1008 30.7.3 Import of Precursor Proteins Unfolded With Urea 1009 30.7.4 Kinetic Analysis of the Unfolding Reaction by Trapping of Intermediates 1009 References 1011 31 The Chaperone System of Mitochondria Wolfgang Voos and Nikolaus Pfanner 1020 31.1 Introduction 1020 31.2 Membrane Translocation and the Hsp70 Import Motor 1020 31.3 Folding of Newly Imported Proteins Catalyzed by the Hsp70 and Hsp60 Systems 1026 31.4 Mitochondrial Protein Synthesis and the Assembly Problem 1030 31.5 Aggregation versus Degradation: Chaperone Functions Under Stress Conditions 1033 31.6 Experimental Protocols 1034 31.6.1 Chaperone Functions Characterized With Yeast Mutants 1034 31.6.2 Interaction of Imported Proteins With Matrix Chaperones 1036 31.6.3 Folding of Imported Model Proteins 1037 31.6.4 Assaying Mitochondrial Degradation of Imported Proteins 1038 31.6.5 Aggregation of Proteins in the Mitochondrial Matrix 1038 References 1039 32 Chaperone Systems in Chloroplasts Thomas Becker, Ju¨rgen Soll, and Enrico Schleiff 1047 32.1 Introduction 1047 32.2 Chaperone Systems within Chloroplasts 1048 32.2.1 The Hsp70 System of Chloroplasts 1048 32.2.1.1 The Chloroplast Hsp70s 1049 32.2.1.2 The Co-chaperones of Chloroplastic Hsp70s 1051 32.2.2 The Chaperonins 1052 32.2.3 The HSP100/Clp Protein Family in Chloroplasts 1056 32.2.4 The Small Heat Shock Proteins 1058 32.2.5 Hsp90 Proteins of Chloroplasts 1061 32.2.6 Chaperone-like Proteins 1062 32.2.6.1 The Protein Disulfide Isomerase (PDI) 1062 32.2.6.2 The Peptidyl-prolyl cis Isomerase (PPIase) 1063 32.3 The Functional Chaperone Pathways in Chloroplasts 1065 32.3.1 Chaperones Involved in Protein Translocation 1065 32.3.2 Protein Transport Inside of Plastids 1070 32.3.3 Protein Folding and Complex Assembly Within Chloroplasts 1071 32.3.4 Chloroplast Chaperones Involved in Proteolysis 1072 32.3.5 Protein Storage Within Plastids 1073 32.3.6 Protein Protection and Repair 1074 32.4 Experimental Protocols 1075 32.4.1 Characterization of Cpn60 Binding to the Large Subunit of Rubisco via Native PAGE (adopted from Ref. [6]) 1075 32.4.2 Purification of Chloroplast Cpn60 From Young Pea Plants (adopted from Ref. [203]) 1076 32.4.3 Purification of Chloroplast Hsp21 From Pea (Pisum sativum) (adopted from [90]) 1077 32.4.4 Light-scattering Assays for Determination of the Chaperone Activity Using Citrate Synthase as Substrate (adopted from [196]) 1078 32.4.5 The Use Of Bis-ANS to Assess Surface Exposure of Hydrophobic Domains of Hsp17 of Synechocystis (adopted from [202]) 1079 32.4.6 Determination of Hsp17 Binding to Lipids (adopted from Refs. [204, 205]) 1079 References 1081 33 An Overview of Protein Misfolding Diseases Christopher M. Dobson 1093 33.1 Introduction 1093 33.2 Protein Misfolding and Its Consequences for Disease 1094 33.3 The Structure and Mechanism of Amyloid Formation 1097 33.4 A Generic Description of Amyloid Formation 1101 33.5 The Fundamental Origins of Amyloid Disease 1104 33.6 Approaches to Therapeutic Intervention in Amyloid Disease 1106 33.7 Concluding Remarks 1108 Acknowledgements 1108 References 1109 34 Biochemistry and Structural Biology of Mammalian Prion Disease Rudi Glockshuber 1114 34.1 Introduction 1114 34.1.1 Prions and the ``Protein-Only'' Hypothesis 1114 34.1.2 Models of PrPSc Propagation 1115 34.2 Properties of PrPC and PrPSc 1117 34.3 Three-dimensional Structure and Folding of Recombinant PrP 1120 34.3.1 Expression of the Recombinant Prion Protein for Structural and Biophysical Studies 1120 34.3.2 Three-dimensional Structures of Recombinant Prion Proteins from Different Species and Their Implications for the Species Barrier of Prion Transmission 1120 34.3.2.1 Solution Structure of Murine PrP 1120 34.3.2.2 Comparison of Mammalian Prion Protein Structures and the Species Barrier of Prion Transmission 1124 34.3.3 Biophysical Characterization of the Recombinant Prion Protein 1125 34.3.3.1 Folding and Stability of Recombinant PrP 1125 34.3.3.2 Role of the Disulfide Bond in PrP 1127 34.3.3.3 Influence of Point Mutations Linked With Inherited TSEs on the Stability of Recombinant PrP 1129 34.4 Generation of Infectious Prions in Vitro: Principal Difficulties in Proving the Protein-Only Hypothesis 1131 34.5 Understanding the Strain Phenomenon in the Context of the Protein-Only Hypothesis: Are Prions Crystals? 1132 34.6 Conclusions and Outlook 1135 34.7 Experimental Protocols 1136 34.7.1 Protocol 1 [53, 55] 1136 34.7.2 Protocol 2 [54] 1137 References 1138 35 Insights into the Nature of Yeast Prions Lev Z. Osherovich and Jonathan S. Weissman 1144 35.1 Introduction 1144 35.2 Prions as Heritable Amyloidoses 1145 35.3 Prion Strains and Species Barriers: Universal Features of Amyloid-based Prion Elements 1149 35.4 Prediction and Identification of Novel Prion Elements 1151 35.5 Requirements for Prion Inheritance beyond Amyloid-mediated Growth 1154 35.6 Chaperones and Prion Replication 1157 35.7 The Structure of Prion Particles 1158 35.8 Prion-like Structures as Protein Interaction Modules 1159 35.9 Experimental Protocols 1160 35.9.1 Generation of Sup35 Amyloid Fibers in Vitro 1160 35.9.2 Thioflavin T--based Amyloid Seeding Efficacy Assay (Adapted from Chien et al. 2003) 1161 35.9.3 AFM-based Single-fiber Growth Assay 1162 35.9.4 Prion Infection Protocol (Adapted from Tanaka et al. 2004) 1164 35.9.5 Preparation of Lyticase 1165 35.9.6 Protocol for Counting Heritable Prion Units (Adapted from Cox et al. 2003) 1166 Acknowledgements 1167 References 1168 36 Polyglutamine Aggregates as a Model for Protein-misfolding Diseases Soojin Kim, James F. Morley, Anat Ben-Zvi, and Richard I. Morimoto 1175 36.1 Introduction 1175 36.2 Polyglutamine Diseases 1175 36.2.1 Genetics 1175 36.2.2 Polyglutamine Diseases Involve a Toxic Gain of Function 1176 36.3 Polyglutamine Aggregates 1176 36.3.1 Presence of the Expanded Polyglutamine Is Sufficient to Induce Aggregation in Vivo 1176 36.3.2 Length of the Polyglutamine Dictates the Rate of Aggregate Formation 1177 36.3.3 Polyglutamine Aggregates Exhibit Features Characteristic of Amyloids 1179 36.3.4 Characterization of Protein Aggregates in Vivo Using Dynamic Imaging Methods 1180 36.4 A Role for Oligomeric Intermediates in Toxicity 1181 36.5 Consequences of Misfolded Proteins and Aggregates on Protein Homeostasis 1181 36.6 Modulators of Polyglutamine Aggregation and Toxicity 1184 36.6.1 Protein Context 1184 36.6.2 Molecular Chaperones 1185 36.6.3 Proteasomes 1188 36.6.4 The Protein-folding ``Buffer'' and Aging 1188 36.6.5 Summary 1189 36.7 Experimental Protocols 1190 36.7.1 FRAP Analysis 1190 References 1192 37 Protein Folding and Aggregation in the Expanded Polyglutamine Repeat Diseases Ronald Wetzel 1200 37.1 Introduction 1200 37.2 Key Features of the Polyglutamine Diseases 1201 37.2.1 The Variety of Expanded PolyGln Diseases 1201 37.2.2 Clinical Features 1201 37.2.2.1 Repeat Expansions and Repeat Length 1202 37.2.3 The Role of PolyGln and PolyGln Aggregates 1203 37.3 PolyGln Peptides in Studies of the Molecular Basis of Expanded Polyglutamine Diseases 1205 37.3.1 Conformational Studies 1205 37.3.2 Preliminary in Vitro Aggregation Studies 1206 37.3.3 In Vivo Aggregation Studies 1206 37.4 Analyzing Polyglutamine Behavior With Synthetic Peptides: Practical Aspects 1207 37.4.1 Disaggregation of Synthetic Polyglutamine Peptides 1209 37.4.2 Growing and Manipulating Aggregates 1210 37.4.2.1 Polyglutamine Aggregation by Freeze Concentration 1210 37.4.2.2 Preparing Small Aggregates 1211 37.5 In vitro Studies of PolyGln Aggregation 1212 37.5.1 The Universe of Protein Aggregation Mechanisms 1212 37.5.2 Basic Studies on Spontaneous Aggregation 1213 37.5.3 Nucleation Kinetics of PolyGln 1215 37.5.4 Elongation Kinetics 1218 37.5.4.1 Microtiter Plate Assay for Elongation Kinetics 1219 37.5.4.2 Repeat-length and Aggregate-size Dependence of Elongation Rates 1220 37.6 The Structure of PolyGln Aggregates 1221 37.6.1 Electron Microscopy Analysis 1222 37.6.2 Analysis with Amyloid Dyes Thioflavin T and Congo Red 1222 37.6.3 Circular Dichroism Analysis 1224 37.6.4 Presence of a Generic Amyloid Epitope in PolyGln Aggregates 1225 37.6.5 Proline Mutagenesis to Dissect the Polyglutamine Fold Within the Aggregate 1225 37.7 Polyglutamine Aggregates and Cytotoxicity 1227 37.7.1 Direct Cytotoxicity of PolyGln Aggregates 1228 37.7.1.1 Delivery of Aggregates into Cells and Cellular Compartments 1229 37.7.1.2 Cell Killing by Nuclear-targeted PolyGln Aggregates 1229 37.7.2 Visualization of Functional, Recruitment-positive Aggregation Foci 1230 37.8 Inhibitors of polyGln Aggregation 1231 37.8.1 Designed Peptide Inhibitors 1231 37.8.2 Screening for Inhibitors of PolyGln Elongation 1231 37.9 Concluding Remarks 1232 37.10 Experimental Protocols 1233 37.10.1 Disaggregation of Synthetic PolyGln Peptides 1233 37.10.2 Determining the Concentration of Low-molecular-weight PolyGln Peptides by HPLC 1235 Acknowledgements 1237 References 1238 38 Production of Recombinant Proteins for Therapy, Diagnostics, and Industrial Research by in Vitro Folding Christian Lange and Rainer Rudolph 1245 38.1 Introduction 1245 38.1.1 The Inclusion Body Problem 1245 38.1.2 Cost and Scale Limitations in Industrial Protein Folding 1248 38.2 Treatment of Inclusion Bodies 1250 38.2.1 Isolation of Inclusion Bodies 1250 38.2.2 Solubilization of Inclusion Bodies 1250 38.3 Refolding in Solution 1252 38.3.1 Protein Design Considerations 1252 38.3.2 Oxidative Refolding With Disulfide Bond Formation 1253 38.3.3 Transfer of the Unfolded Proteins Into Refolding Buffer 1255 38.3.4 Refolding Additives 1257 38.3.5 Cofactors in Protein Folding 1260 38.3.6 Chaperones and Folding-helper Proteins 1261 38.3.7 An Artificial Chaperone System 1261 38.3.8 Pressure-induced Folding 1262 38.3.9 Temperature-leap Techniques 1263 38.3.10 Recycling of Aggregates 1264 38.4 Alternative Refolding Techniques 1264 38.4.1 Matrix-assisted Refolding 1264 38.4.2 Folding by Gel Filtration 1266 38.4.3 Direct Refolding of Inclusion Body Material 1267 38.5 Conclusions 1268 38.6 Experimental Protocols 1268 38.6.1 Protocol 1: Isolation of Inclusion Bodies 1268 38.6.2 Protocol 2: Solubilization of Inclusion Bodies 1269 38.6.3 Protocol 3: Refolding of Proteins 1270 Acknowledgements 1271 References 1271 39 Engineering Proteins for Stability and Efficient Folding Bernhard Schimmele and Andreas Plückthun 1281 39.1 Introduction 1281 39.2 Kinetic and Thermodynamic Aspects of Natural Proteins 1281 39.2.1 The Stability of Natural Proteins 1281 39.2.2 Different Kinds of ``Stability'' 1282 39.2.2.1 Thermodynamic Stability 1283 39.2.2.2 Kinetic Stability 1285 39.2.2.3 Folding Efficiency 1287 39.3 The Engineering Approach 1288 39.3.1 Consensus Strategies 1288 39.3.1.1 Principles 1288 39.3.1.2 Examples 1291 39.3.2 Structure-based Engineering 1292 39.3.2.1 Entropic Stabilization 1294 39.3.2.2 Hydrophobic Core Packing 1296 39.3.2.3 Charge Interactions 1297 39.3.2.4 Hydrogen Bonding 1298 39.3.2.5 Disallowed Phi-Psi Angles 1298 39.3.2.6 Local Secondary Structure Propensities 1299 39.3.2.7 Exposed Hydrophobic Side Chains 1299 39.3.2.8 Inter-domain Interactions 1300 39.3.3 Case Study: Combining Consensus Design and Rational Engineering to Yield Antibodies with Favorable Biophysical Properties 1300 39.4 The Selection and Evolution Approach 1305 39.4.1 Principles 1305 39.4.2 Screening and Selection Technologies Available for Improving Biophysical Properties 1311 39.4.2.1 In Vitro Display Technologies 1313 39.4.2.2 Partial in Vitro Display Technologies 1314 39.4.2.3 In Vivo Selection Technologies 1315 39.4.3 Selection for Enhanced Biophysical Properties 1316 39.4.3.1 Selection for Solubility 1316 39.4.3.2 Selection for Protein Display Rates 1317 39.4.3.3 Selection on the Basis of Cellular Quality Control 1318 39.4.4 Selection for Increased Stability 1319 39.4.4.1 General Strategies 1319 39.4.4.2 Protein Destabilization 1319 39.4.4.3 Selections Based on Elevated Temperature 1321 39.4.4.4 Selections Based on Destabilizing Agents 1322 39.4.4.5 Selection for Proteolytic Stability 1323 39.5 Conclusions and Perspectives 1324 Acknowledgements 1326 References 1326 Index 1334   Bestellen Online-Ausgabe Inhaltsverzeichnis Kurzbeschreibung Langtext Besprechungen Autoreninformation Sitz der Autoren Protein Engineering Handbook Protein Degradation Series 4 Volume Set The Protein Chart [mehr >>] Angewandte Chemie Angewandte Chemie International Edition Chemie Ingenieur Technik [mehr>>] Christie, Daniel J. (ed.) The Encyclopedia of Peace Psychology 385,- Euro gültig bis 31. März 2012 [mehr Angebote >>]

Seite empfehlen          RSS-Feeds             Druckversion ©2012 Wiley-VCH Verlag GmbH & Co. KGaA - Betreiber http://www.wiley-vch.de - mailto: info@wiley-vch.de Datenschutz