Protein Folding: Methods and Protocols

Preface
Contents
Contributors
Part I: Protein Engineering and Protein Chemistry Methods
Chapter 1: Mutational Analysis of Protein Folding Transition States: Phi Values
1 Introduction
2 Materials
3 Methods and Analysis
3.1 Thermodynamic and Kinetic Conventions for 훟 Value Analysis
3.2 Interpretation of the 훟 Value
3.3 Design of Mutations
3.4 Tests to Confirm the Conservation of the Native Structure
3.5 Equilibrium Denaturation Experiments
3.5.1 Experimental Setup
3.5.2 General Protocol
3.6 Kinetic Chevron Experiments
3.6.1 Experimental Setup
3.6.2 General Protocol
3.7 Applications of the 훟 Value Analysis
3.7.1 Folding Transition State Characterization
3.7.2 Experimental Errors, Energetic Perturbations, and Average 훟 Values
3.7.3 Leffler Analysis: Multiple Mutations at the Same Position
4 Notes
References
Chapter 2: Engineered Metal-Binding Sites to Probe Protein Folding Transition States: Psi Analysis
1 Introduction
2 Materials
3 Methods and Analysis
3.1 Derivation of ψ
3.1.1 Models of ψ
3.1.2 Interpretation of ψ0 Values
3.2 Designing biHis Sites
3.3 Equilibrium Denaturation
3.3.1 General Protocol
3.3.2 Application of Equilibrium Data
3.4 Kinetic Chevron Analysis
3.4.1 General Experimental Protocol
3.4.2 Applications and Interpretation
3.4.3 Testing for Fast Ion Binding Equilibrium
3.5 Metal-Dependent Folding Kinetics: Leffler Plot
3.5.1 Analysis of Leffler Plot
Metal Stabilization
Obtaining ψ Values from the Leffler Plot
3.5.2 Delineation Between the Heterogeneous and Homogeneous TSE Scenarios
Cross-link biCys Sites
Measure the Effects of Cross-link
3.6 Comparing ψ0 Values to Simulation Data
3.7 Applications of ψ Beyond Protein Folding
4 Notes
References
Chapter 3: Site-Specific Interrogation of Protein Structure and Stability
1 Introduction
2 Materials
3 Methods
3.1 Site-Specific IR Probes
3.1.1 Site-Specific CD Probes
3.2 Site-Specific Fluorescence Probes
4 Notes
References
Chapter 4: Purification and Handling of the Chaperonin GroEL
1 Introduction
2 Materials
2.1 Chromatographic Purification of GroEL
2.2 Acetone Treatment to Further Purify GroEL
2.3 Coupled-Enzyme ATPase Assay
3 Methods
3.1 Column Chromatographic Purification of GroEL
3.2 Acetone Treatment of Chromatographic Purified GroEL Sample (See Note 4)
3.3 GroEL Purity Assessed by Tryptophan Fluorescence
3.4 GroEL ATPase Activity Measured by Coupled-Enzyme Assay
4 Notes
References
Part II: Kinetic and Thermodynamic Analysis of Protein Folding
Chapter 5: Folding Free Energy Surfaces from Differential Scanning Calorimetry
1 Introduction
2 Materials
3 Methods
3.1 Buffer Preparation
3.2 Protein Preparation
3.3 DSC Experiment (See Note 5)
3.4 Absolute Heat Capacity Calculations
3.5 Data Analysis: Estimation of the Thermodynamic Free Energy Barrier
4 Notes
References
Chapter 6: Fast-Folding Kinetics Using Nanosecond Laser-Induced Temperature-Jump Methods
1 Introduction
1.1 Infrared Absorption
1.2 Fluorescence Spectroscopy
2 Materials
2.1 Instrumentation
2.2 Chemicals and Reagents
2.3 Other Materials
3 Methods
3.1 Sample Preparation for Infrared Absorption Measurements
3.2 Sample Preparation for Fluorescence Measurements
3.3 Infrared Temperature-Jump Kinetic Measurements
3.4 Fluorescence Temperature-Jump Kinetic Measurements
4 Notes
References
Chapter 7: Measurement of Submillisecond Protein Folding Using Trp Fluorescence and Photochemical Oxidation
1 Introduction
2 Materials
3 Methods
3.1 Mixer Fabrication
3.2 Setup
3.3 Aligning Equipment
3.4 Taking Fluorescence Data
3.5 FPOP
3.6 Analyzing with Mass Spec
4 Notes
References
Chapter 8: Native State Hydrogen Exchange-Mass Spectrometry Methods to Probe Protein Folding and Unfolding
1 Introduction
2 Materials
2.1 Buffers for the HX Reaction
2.2 Sample Desalting
2.3 LC-MS
2.4 ETD
3 Methods
3.1 Deuteration of the Protein
3.2 Hydrogen Exchange (HX) Reaction
3.3 Mass Spectrometry of the Intact Protein
3.4 Electron Transfer Dissociation (ETD)
4 Notes
References
Chapter 9: Multi-Probe Equilibrium Analysis of Gradual (Un)Folding Processes
1 Introduction
2 Materials
2.1 Peptide Synthesis
2.2 FTIR Spectroscopy
3 Methods
3.1 Peptide Synthesis
3.2 Post-Synthesis Work-Up and Cleavage
3.3 FTIR Sample Preparation
3.4 FTIR Spectroscopy
3.5 FTIR Data Processing
3.6 Example Data Analysis: Thermal Unfolding
4 Notes
References
Chapter 10: NMR Analysis of Protein Folding Interaction Networks
1 Introduction
2 Materials
2.1 Suitable Proteins
2.1.1 Proteins That (Un)Fold in the Microsecond Timescale
2.1.2 Structural Motifs
2.2 NMR Samples and Experiments
2.2.1 Type of NMR Tubes
2.2.2 Chemicals
2.2.3 Protein Sample
2.2.4 Spectrometer
2.2.5 NMR Experiments
2.2.6 NMR Software
2.3 Computational Analysis
3 Methods
3.1 Conditions Necessary for the Atomic-Resolution Analysis
3.1.1 Reversibility of the Equilibrium Thermal Unfolding
3.1.2 Folding Kinetics in Fast Conformational-Exchange
3.2 NMR Experiments to Monitor Protein Unfolding at Atomic Resolution
3.2.1 Temperature Calibration of the NMR Probe
3.2.2 Chemical Shift Referencing
3.2.3 Chemical Shift Assignment
3.2.4 Monitoring NMR Signal Changes with Temperature
3.3 Analysis of Individual Atomic Equilibrium Unfolding Curves
3.3.1 Classification of Atomic Unfolding Curves
3.3.2 Analysis of Two-State-Like Unfolding Curves
3.3.3 Analysis of Three-State-Like Unfolding Curves
3.4 Clustering of Atomic Unfolding Curves and Network Analysis
3.4.1 Average Atomic Unfolding Behavior from NMR Compared to the Global Unfolding Process
3.4.2 Data Clustering
3.4.3 Calculation of the Thermodynamic Coupling Index Matrix (TCI)
3.4.4 Comparing Thermodynamic Coupling Index Matrix and Native Three-Dimensional Structure
4 Notes
References
Chapter 11: NMR Relaxation Dispersion Methods for the Structural and Dynamic Analysis of Quickly Interconverting, Low-Populate...
1 Introduction
2 Materials
2.1 NMR Samples
2.2 NMR Tubes
3 Methods
3.1 Theory of Relaxation Dispersion NMR
3.1.1 CPMG Relaxation: The Pulse Sequence
3.1.2 RD Experiments: Mathematical Description
3.1.3 Determining the Exchange Parameters by Fitting CPMG Relaxation Dispersion Curves
3.2 Relaxation Dispersion in the Rotating Frame
3.3 Basic NMR Setup
3.3.1 Temperature Calibration of the NMR Probe
3.3.2 15N and 3C Hard Pulse Calibration
3.3.3 Chemical Shift Referencing
3.3.4 Chemical Shift Assignments
3.4 The RD-NMR Experiment
3.4.1 Setting Up an RD-NMR Experiment
3.4.2 Data Analysis
4 Notes
References
Part III: Single-Molecule Spectroscopy Techniques
Chapter 12: Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy
1 Introduction
2 Materials
2.1 Instruments
2.2 Reagents
3 Methods
3.1 Selection of Labeling Sites and Fluorophores
3.1.1 Design Considerations for Introducing Cysteine Residues
3.1.2 Choice of Dye Pair
3.2 Preparing the Protein for Labeling
3.2.1 RP-HPLC-Based Purification
3.2.2 Ion-Exchange-Based Purification
3.3 Labeling Reaction
3.4 Purification of Dye-Labeled Protein
3.4.1 RP-HPLC-Based Purification
3.4.2 Ion-Exchange Chromatography-Based Purification
3.5 Preparation of Samples for Single-Molecule Spectroscopy
3.6 Quality Control
3.6.1 Protein Identity and Impurities
3.6.2 UV-Vis Absorption Spectrum
3.6.3 Single-Molecule Spectroscopy
3.6.4 Site-Specific Labeling
3.6.5 Methionine Oxidation
4 Notes
References
Chapter 13: Single-Molecule Fluorescence Spectroscopy Approaches for Probing Fast Biomolecular Dynamics and Interactions
1 Introduction
2 Materials
2.1 Confocal Microscope
2.2 Photoprotection Cocktail
2.3 Sample Well
3 Methods
3.1 Optimizing Confocal Microscope Setup for High Count Rates
3.1.1 Setup of the Confocal Microscope for High Count Rates
3.1.2 Alignment of the Excitation Pathway
3.1.3 Alignment of the Emission Pathway
3.2 Preparation of the Photoprotection Cocktail
3.3 Preparation of the Sample Well for Small Volume Samples
3.3.1 Cleaning the Sample Holder and Coverslips
3.3.2 PEGylation of the Sample Holder
3.3.3 Long-Term Storage
3.4 Single-Molecule Fluorescence Measurements with the Confocal Microscope
4 Notes
References
Chapter 14: Theory and Analysis of Single-Molecule FRET Experiments
1 Introduction
2 Photon Statistics and Basic Parameters
2.1 Photon Count Rates
2.2 Apparent FRET Efficiency
3 Methods
3.1 FRET Efficiency Histograms
3.1.1 Single Conformational State
3.1.2 Multiple Non-interconverting Conformational States
3.1.3 Multiple States with Conformational Dynamics
3.1.4 Two-State Dynamics
3.1.5 Gaussian Approximation
3.2 Likelihood-Based Analysis of Photon Sequences
3.2.1 Single Conformational State
3.2.2 Multiple Conformations
3.2.3 Reduced Likelihood
3.2.4 Non-interconverting Species
3.2.5 Accuracy of the Parameter Estimation
3.3 Recoloring Photon Sequences
3.4 Detection of Invisible States Using Likelihood Method and Recoloring
3.5 Practical Issues in the Analysis of Free-Diffusion and Immobilization Data
4 Notes
References
Chapter 15: Mechanochemical Evolution of Disulfide Bonds in Proteins
1 Introduction
2 Materials
2.1 Phylogenetic Analysis and ASR
2.2 Protein Cloning, Expression, Purification, and Oxidation
2.2.1 Protein Plasmid (See Note 1)
2.2.2 Protein Cloning, Transformation, and Amplification
2.2.3 Protein Expression and Purification (See Note 2)
2.3 Preparation of AFS Gold Substrates
2.4 SmFS-AFS Experiments
2.5 Data Analysis of smFS-AFS Experiments
3 Methods
3.1 Phylogenetic Analysis and ASR
3.2 Protein Cloning, Expression, Purification, and Oxidation
3.2.1 Cloning
3.2.2 Screening Expression Test
3.2.3 Large-Scale Expression
3.2.4 Purification (See Note 3)
3.3 Preparation of AFS Gold Substrates
3.4 SmFS-AFS Experiments (See Notes 4 and 5)
3.5 Data Analysis of smFS-AFS Experiments
3.5.1 Force-Extension Data Analysis
3.5.2 Force-Clamp Data Analysis
4 Notes
References
Part IV: Molecular Simulations
Chapter 16: Coarse-Grained Simulations of Protein Folding: Bridging Theory and Experiments
1 Introduction
2 Methods
2.1 Structure-Based Models (SBM): Potential and Cα Model
2.2 Data Analysis and Connection to Experiments
2.2.1 Thermodynamics
Folding Temperature and Free Energy Profile
훟-Value Analysis
Folding Route
2.3 Kinetics
2.3.1 Folding Times and Diffusion
2.3.2 Non-native Interactions and Frustration
3 Conclusions
References
Chapter 17: Analysis of Molecular Dynamics Simulations of Protein Folding
1 Introduction
2 Materials
3 Methods
3.1 Identifying or Optimizing Reaction Coordinates for Describing Folding
3.1.1 Identify Trial Coordinates for Testing
3.1.2 Define Unfolded and Folded States in Terms of Given Reaction Coordinates
3.1.3 Define Transition Paths Between Unfolded and Folded
3.1.4 Compute Histograms of Coordinate from Equilibrium Folding Trajectories
3.1.5 Quantifying and Optimizing Reaction Coordinates
3.2 Identifying Key Interactions Which Determine Folding Mechanism
3.2.1 Compute Contact Maps for Entire Trajectory
3.2.2 Compute Contact Map Averages
3.2.3 Calculate Bayesian Criterion for Contacts
3.2.4 Alternative Measure: Contact Lifetimes
3.2.5 Determining Contact Lifetimes
3.3 Comparing with Experiment: Computing 훟-Values
3.3.1 Computing 훟i from Long MD Simulations from Putative Transition States
3.3.2 Computing 훟i from Long MD Simulations from Long Trajectories Using Transition Path Theory (TPT)
3.3.3 Computing 훟i from Long MD Simulations from Long Trajectories Using Folding Flux (FF)
4 Notes
References
Chapter 18: Atomistic Simulations of Thermal Unfolding
1 Introduction
2 Performing Replica Exchange Molecular Dynamics Simulations
2.1 Practical Issues When Implementing REMD
2.1.1 Sampling from a Canonical Ensemble
2.1.2 Determining the Temperature Distribution
2.1.3 Sampling Efficiency
2.1.4 Exchange Attempt Frequency and Exchange Rates
2.1.5 Selection of Force Fields
2.1.6 Reaching ``Equilibrium´´
3 Calculation of Thermodynamic Properties from REMD Simulations
3.1 Data Analysis and Quantification of Errors
3.2 Block Averages
References
Chapter 19: Molecular Simulations of Intrinsically Disordered Proteins and Their Binding Mechanisms
1 Introduction
2 Materials
3 Methods
3.1 The Protein Model for Simulations
3.2 All-Atom Molecular Dynamics Simulations
3.2.1 Input Protein Structure
3.2.2 Generating the Topology File
3.2.3 System Preparation
3.2.4 Simulation Protocols
3.2.5 Equilibration Simulation
3.2.6 Production Molecular Dynamics Simulation
3.2.7 Trajectory Analysis
3.3 Molecular Simulations with Structure-Based Models
3.3.1 A Cα Structure-Based Model
The Structural Representation
Potentials
3.3.2 Structure-Based Model Simulation Procedures
3.3.3 Empirical Parameterization of the Structured-Based Model
Electrostatic Interactions
Temperature
pKID Intrachain Interactions
pKID-KIX Interchain Interactions
3.3.4 Production Coarse-Grained Simulation
3.3.5 Analysis
4 Notes
References
Part V: Prediction Methods
Chapter 20: Prediction of Folding and Unfolding Rates of Proteins with Simple Models
1 Introduction
2 Theory
2.1 Thermodynamics
2.2 Kinetics
3 Methods
3.1 Splitting Stabilization Energy in Local and Non-local Contributions
3.2 Parametrization
3.3 Prediction
3.4 The PREFUR Package
4 Notes
References
Chapter 21: Predicting and Simulating Mutational Effects on Protein Folding Kinetics
1 Introduction
2 Materials
2.1 Reaction Coordinate
2.2 Conformational Entropy and Enthalpy
2.3 One-Dimensional Free Energy Profile
2.4 Calculating Stabilities and Free Energy Barrier Heights
3 Methods
3.1 Simulating Diffusive Kinetics
3.2 Reproducing Wild-Type Stability, Relaxation Rate, and Simulating WT Free Energy Profiles
3.3 Simulating Mutant Free Energy Profiles and Predicting Kinetics
3.4 Φ-Values and the Folding Transition State (TS)
3.5 Estimating Energetic Contributions to Φ-Values from Free Energy Profiles
4 Notes
References
Chapter 22: Localization of Energetic Frustration in Proteins
1 Introduction
2 Materials
2.1 Protein Structure
2.2 Energy Function
2.3 Decoy Set
3 Methods
3.1 Energies of Decoys
3.2 Frustration Index
3.3 Visualizing and Analyzing Results
3.4 Interpreting Results
4 Notes
References
Chapter 23: Modeling the Structure, Dynamics, and Transformations of Proteins with the UNRES Force Field
1 Introduction
2 Methods
2.1 UNRES Model
2.2 UNRES Force Field
2.3 Types of Calculations
2.4 Analysis of UNRES Results
2.5 Programming Language
2.6 Parallelization
2.7 Availability
3 Applications
3.1 Protein Folding Pathways and Kinetics
3.2 Determination of Most Likely Conformations (Protein-Structure Prediction)
3.3 Examples of Applications to Biological Problems
References
Index