Protein-ligand interactions : methods and applications

Description

Preface Contents Contributors Part I: Overview Chapter 1: Assessing and Improving Protein Sample Quality 1 Introduction 1.1 Preassessment of Purity and Concentration Determination by Ultraviolet Spectroscopy 1.2 Assessing Protein Purity, Homogeneity, and Oligomeric State 1.2.1 Assessing Purity 1.2.2 Assessing Homogeneity 1.2.3 Assessing Identity and Chemical Integrity 1.3 Assessing Structural Integrity 1.4 Assessing Protein Stability and Solubility 1.4.1 Thermal Unfolding Assays 1.4.2 Assessing Colloidal Stability/Aggregation 1.4.3 Buffer Optimization 1.4.4 Storage Issues 1.4.5 Membrane Protein Buffer Optimization 1.5 Batch-to-Batch Consistency 2 Materials and Methods 2.1 UV Spectrum and Concentration Determination 2.2 Methods to Determine Purity, Homogeneity, and Oligomeric State 2.2.1 SDS-PAGE and Staining Polyacrylamide Gels Discontinuous Denaturing SDS-PAGE Staining 2.2.2 Size-Exclusion Chromatography: Static Light Scattering (SEC-LALS/SEC-MALS) 2.2.3 DLS Measurement of Homogeneity 2.2.4 Protein Intact Mass by MALDI-TOF Mass Spectrometry 2.3 Protein Structural Integrity and Stability 2.3.1 CD Spectroscopy Choice of Cuvette, Buffer, and Sample Preparation Spectrometer Setup Measurement of a CD Spectrum Estimation of Secondary Structure Composition Determination of Protein Thermostability Information About the Tertiary Structure 2.3.2 DSF of Intrinsic Fluorophores 2.3.3 Thermofluor/DSF of Extrinsic Fluorophores 2.3.4 DSC 2.4 Buffer Optimization 3 Notes References Chapter 2: A Familiar Protein-Ligand Interaction Revisited with Multiple Methods 1 Introduction 1.1 Several Techniques, One Experimental System 1.2 A Brief Introduction to HEWL and NAG3 1.3 A Brief Introduction to the Techniques Used 1.4 Nature and Analysis of Example Data 2 Materials 2.1 Buffers 2.2 Protein and Ligand 2.3 Other Reagents 2.4 Instruments 3 Methods 3.1 Preparation of Protein and Ligand Solutions 3.1.1 Preparation of HEWL Solution 3.1.2 Preparation of NAG3 Solution 3.2 Thermal Shift Assay 3.2.1 Thermal Shift Assay Measurement 3.2.2 Thermal Shift Assay Data Analysis and Typical Results 3.3 Fluorescence Intensity 3.3.1 Preparation of Titration for Fluorescence Intensity 3.3.2 Fluorescence Intensity Measurement 3.3.3 Fluorescence Intensity Data Analysis and Typical Results 3.4 Microscale Thermophoresis (MST) 3.4.1 Preparation of Fluorescently Labeled HEWL for MST 3.4.2 Preparation of Titration for MST 3.4.3 MST Measurement 3.4.4 MST Data Analysis and Typical Results 3.5 Isothermal Titration Calorimetry (ITC) 3.5.1 ITC Measurement 3.5.2 ITC Data Analysis and Typical Results 3.6 SPR 3.6.1 Immobilization of HEWL 3.6.2 SPR Measurement 3.6.3 SPR Data Analysis and Typical Results 4 Notes References Part II: Universal Methods for Protein Interactions Chapter 3: Interactions of a Signal Transduction Protein Investigated by Fluorescence Stopped-Flow Kinetics 1 Introduction 1.1 The Biological System Under Study 1.2 The Choice of Techniques 1.3 Reaction Kinetics and Thermodynamics 2 Materials 2.1 Instrumentation 2.2 Instrument Settings 2.3 Samples Used in This Study 3 Methods 3.1 Simple Bimolecular Binding Reactions 3.1.1 Results and Common Problems 3.1.2 Alternative Approach 3.2 Competitive Binding 3.2.1 Measuring k-1 Using an Excess of N to Compete Labeled L from PL 3.2.2 Measuring the Association Rate Constant for N Binding to P 3.3 Ternary Complex Formation 3.4 Multistep Reactions 3.5 Data Analysis and Simulation 4 Notes References Chapter 4: Kinetic Methods of Deducing Binding Mechanisms Involving Intrinsically Disordered Proteins 1 Introduction 1.1 Intrinsically Disordered Proteins 1.2 A Kinetic Approach to Assess Mechanism 2 Materials 2.1 The Stopped-Flow Instrument 2.2 Protein Samples 2.3 Buffers 3 Methods 3.1 Designing and Performing Binding Experiments 3.2 Dealing with Second-Order Conditions 3.3 Three State Binding Reactions 3.4 Displacement Experiments to Determine koff 3.5 Further Control Experiments and Common Artifacts 3.6 Example Studies and What They Tell Us About IDP Binding 3.6.1 Case Study 1: p53TAD and MDM2, an Apparent One-Step Binding 3.6.2 Case Study 2: NTAIL and XD Domain, a Two-Step Binding 3.6.3 Case Study 3: ACTR and NCBD, Multistep Binding with Several Kinetic Phases 3.6.4 Case Study 4: HPV E7 and Rb, a Multistep Binding with Several Kinetic Phases 3.6.5 Case Study 5: Using Linear Free Energy Relationships to Access the Overall Properties of the Transition State for Bindin... 4 Notes References Chapter 5: Isothermal Titration Calorimetry 1 Introduction 1.1 ITC: A Measurement Nirvana? 1.2 Why Read This Chapter? 2 Materials 2.1 ITC Instrumental Basics 2.2 A Realistic Test Reaction: Lysozyme Binding a Simple Trisaccharide Ligand 3 Methods 3.1 Running the Test Reaction 3.2 Data Inspection and Fitting 3.3 Are the Concentrations of Protein and Ligand Optimal? 3.4 How Do the Concentrations Influence the Results? 3.5 Occam´s Razor and the Use of Advanced Data Fitting 3.6 Titrations Either Way Round: Varying Ligand or Protein 3.7 All Heat Looks the Same: There Are No Different ``Colors´´ 3.8 Measurement and Temperature: The Heat Capacity for Binding 3.9 Same ``Color´´ of Heat, But Different Kinetics 3.10 Not Just Protein-Ligand Interactions 4 Notes References Chapter 6: Measuring the KD of Protein-Ligand Interactions Using Microscale Thermophoresis 1 Introduction 2 Materials 2.1 Buffers/Detergent 2.2 Proteins 2.3 Dye Labeling 2.4 Thermophoresis Supplies 2.5 Software/Computer 2.6 Instrumentation 3 Methods 3.1 Labeling the Receptor 3.2 Optimizing Capillaries, LED Power, and Buffer Conditions 3.3 Optimizing MST Power 3.4 Performing the MST Experiment 3.5 Data Analysis 3.6 Saving and Documenting the Analysis 4 Notes References Chapter 7: Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D): Preparing Functionalized Lipid Layers for the Stud... 1 Introduction 2 Materials 2.1 Buffers 2.2 Proteins 2.3 Reagents 3 Method 3.1 Preparation of Multilamellar Vesicles 3.2 Lipid Extrusion to Produce Unilamellar Vesicles (See Note 7) 3.3 Cleaning and Preparation of Qsense SiO2 Sensors (See Note 11) 3.4 Lipid Bilayer Formation Using QCM-D 3.4.1 Mounting Sensor in the E1 System 3.4.2 Lipid Bilayer Deposition and Measurement Procedure 4 Notes References Part III: Screening for Ligand Binding Chapter 8: Indirect Detection of Ligand Binding by Thermal Melt Analysis 1 Introduction 2 Materials 2.1 Optimization of Conditions and Screening of Compounds by DSF Experiments 2.2 Screening of Compounds by DSF 2.3 Quantitative AlphaScreen Assay to Measure Soluble Target Protein for CETSA 2.4 Thermal Melt Analysis in Live Cells by CETSA 2.5 Quantification of Thermostable Target Protein by AlphaScreen 2.6 Screening of Compounds at a Single Optimized Temperature by CETSA HT 3 Methods 3.1 Optimization of Conditions for DSF Experiments 3.2 Screening of Compounds by DSF 3.3 Quantitative AlphaScreen Assay to Measure Soluble Target Protein for CETSA 3.4 Thermal Melt Analysis in Live Cells by CETSA 3.5 Quantification of Thermostable Target Protein by AlphaScreen 3.6 Screening of Compounds at a Single Optimized Temperature by CETSA HT 4 Notes References Chapter 9: The Use of Acoustic Mist Ionization Mass Spectrometry for High-Throughput Screening 1 Introduction 2 Materials 2.1 Instrumentation 2.2 Plates 2.3 Compound Dispensers for Assay-Ready Plate Production 2.4 High-Throughput Platform 2.4.1 Assay Plate Production 2.4.2 Assay Plate Read 2.5 Buffers/Reagents 2.6 Software 3 Method 3.1 Instrument Setup 3.1.1 Spray 3.1.2 Charging 3.1.3 Polarity Switching 3.1.4 Target Enhancement 3.1.5 Buffer Considerations for Acoustic Settings 3.1.6 Detection 3.2 Assay Development for AMI-MS 3.3 Screening 3.4 Automated Screening 3.4.1 Automation of Assay Plate Production 3.4.2 Automation of Assay Plate Read on the AMI-MS 3.5 Instrument Cleaning and Maintenance 3.6 Data Acquisition 3.7 Data Analysis 4 Notes References Chapter 10: Ligand Discovery: High-Throughput Binding: Fluorescence Polarization (Anisotropy) 1 Introduction 2 Materials 3 Methods 3.1 Solution Handling 3.2 Instrumentation and Calibration 3.3 Experimental Design and Assay Development 3.3.1 Measurement of Background Fluorescence 3.3.2 Selection of a Binding Probe 3.3.3 Measurement of the Time to Equilibrium 3.3.4 Effect of Binding on Total Fluorescence 3.3.5 Measurement of Binding of the Probe to the Target 3.3.6 Reagent Stability 3.4 Measuring the Effect of Test Compounds 3.4.1 Calculation of Affinity from IC50 3.4.2 Identifying Compound Interference 4 Notes References Chapter 11: Fragment Screening by NMR 1 Introduction 2 Materials 2.1 NMR Spectrometer and Accessories 2.2 Fragment Library 2.3 Protein 2.4 Tool Compounds 2.5 Analysis Software 3 Methods 3.1 Characterization of the Fragment Library 3.1.1 Sample Preparation 3.1.2 Data Acquisition 3.1.3 Data Analysis 3.2 Characterization of the Target Protein 3.3 Development of NMR Fragment Screening Assay 3.3.1 Binding of a Low-Affinity Tool Compound 3.3.2 Displacement of the Tool Compound by a Potent Competitor 3.3.3 Stability of Binding and Competition 3.3.4 Trial Screen 3.4 Screening the Library 3.5 Data Analysis 3.5.1 1D 1H NMR 3.5.2 STD 3.5.3 Water-LOGSY 3.5.4 Relaxation-Filtered 1D 3.5.5 Combined Data Analysis 3.6 Analysis Software 3.7 Singleton Validation 3.8 Orthogonal Biophysical Validation 4 Notes References Part IV: Nucleotide Binding and Hydrolysis Chapter 12: A Quick Primer in Fluorescence-Based Equilibrium and Pre-steady State Methods for Determining Protein-Nucleotide A... 1 Introduction 2 Materials 2.1 General 2.2 Equilibrium Nucleotide Binding 2.3 Pre-steady State Nucleotide Binding 3 Methods 3.1 Equilibrium Nucleotide Binding 3.1.1 Experimental Procedure 3.1.2 Data Analysis 3.2 Pre-steady State Nucleotide Binding 3.2.1 Experimental Design 3.2.2 Experimental Procedure (See Note 11) 3.2.3 Data Analysis 4 Notes References Chapter 13: Measurement of Nucleotide Hydrolysis Using Fluorescent Biosensors for Phosphate 1 Introduction 2 Materials 2.1 Phosphate Biosensor 2.2 Reaction Buffer 2.3 Nucleotide Solutions 2.4 Pi Standard 2.5 Pi Mop 2.6 Instrumentation 2.7 Data Fitting Software 3 Methods 3.1 Steady State Kinetic Assay to Determine Km and kcat 3.1.1 General Principles 3.1.2 Experimental Design and Optimization Biosensor Concentration Concentration of Substrate Enzyme Concentration Fitting of Initial Rates 3.1.3 Example Protocol: Steady State Kinetic Assay-Chd1 ATPase Buffer and Concentrations Calibration Determine kcat and Km of the Basal and dsDNA-Activated ATPase of Chd1 Testing the Linearity of Measured ATPase Rates with Enzyme Concentration 3.2 Pi Release Kinetics Under Pre-steady State Conditions Using Stopped-Flow 3.2.1 General Principle 3.2.2 Example Protocol: Pi Release Under Single-Turnover Conditions-SufBC ATPase Buffer and Concentrations Stopped-Flow Setup and Cleaning Pi Calibration Pi Release Kinetics Data Interpretation and Analysis 3.3 Extensions and Modification of the Assay 3.4 Summary 4 Notes References Part V: Binding Nucleic Acids Chapter 14: Gel-Based Analysis of Protein-Nucleic Acid Interactions 1 Introduction 2 Materials 2.1 Native Polyacrylamide Gel 2.2 Protein and Nucleic Acid Preparation 2.3 Detection Methods 3 Methods 3.1 Preparation of Polyacrylamide Gel 3.2 Sample Preparation 3.3 Polyacrylamide Gel Electrophoresis 3.4 Gel Imaging 3.5 Variation: Semiquantitative Estimation of Interaction Affinity 3.6 Variation: Supershift of Ternary Protein -Protein-Nucleic Acid Complexes 3.7 Variation: Dual-Color Competition EMSA 4 Notes References Chapter 15: Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE 1 Introduction 2 Materials 2.1 Instruments and Accessories 2.2 Buffers 2.3 Ligand Preparation 2.4 Analyte Preparation 3 Methods 3.1 Ribosome Preparation 3.2 Designing the switchSENSE Experiment 3.2.1 Experimental Considerations 3.2.2 Experimental Workflow Building 3.3 Performing the switchSENSE Experiment 3.4 switchSENSE Data Analysis 4 Notes References Chapter 16: Biolayer Interferometry: Protein-RNA Interactions 1 Introduction 1.1 Protein-RNA Interactions 1.2 Biolayer Interferometry 1.3 Kinetic Theory 2 Materials 2.1 Instrumentation 2.2 Consumables 2.3 Reagents 3 Methods 3.1 Standard Binding Experiment 3.2 Ternary Complexes 3.3 Competition 3.4 Data Analysis 4 Notes References Chapter 17: Analysis of Protein-DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip 1 Introduction 2 Materials 2.1 Buffers and Reagents 2.2 Instrument and Chip and Protein Sample (See Note 2) 2.3 DNA 3 Methods 3.1 Preparation of the ReDCaT Chip 3.2 A General Protocol to Screen for Protein:DNA Interactions 3.3 Analysis of Results 3.4 A Worked Example with Explanation of How Some Typical Results Are Analyzed 3.5 Further Possible Experiments and Uses 4 Notes References Chapter 18: Characterization of Protein-Nucleic Acid Complexes by Size-Exclusion Chromatography Coupled with Light Scattering,... 1 Introduction 1.1 Basic Principles of Light Scattering Measurement 1.2 Light Scattering Coupled with Chromatography 2 Materials 2.1 SEC/MALS System 2.1.1 Instruments (See Note 1) 2.1.2 SEC Column 2.2 Reagents and Supplies 2.3 Sample 3 Methods 3.1 System Setup and Validation 3.2 System Equilibration 3.3 Validation of SEC/MALS System in the Buffer of Interest 3.4 Determination of the Molecular Weight of the Sample Protein, Nucleic Acid, and Protein-Nucleic Acid Complex 3.5 Determination of Monodispersity 3.6 Determination of Stoichiometry, i.e., Protein to Nucleic Acid Ratio in the Complex 4 Notes References Chapter 19: Analytical Ultracentrifugation for Analysis of Protein-Nucleic Acid Interactions 1 Introduction 1.1 Analytical Ultracentrifugation 1.2 Interaction of the Pol III Clamp Loader with SSB-Saturated Template/Primer 1.3 Specific Fluorescence Labeling 1.4 Template/Primer DNA 1.5 Determination of the Affinity of an Interaction by Sedimentation Velocity Experiments 2 Materials 2.1 Buffers and Solutions 2.2 Proteins 2.3 Oligonucleotides 2.4 Peptide 2.5 Instrumentation 2.6 Software 3 Methods 3.1 Labeling of the Clamp Loader 3.2 Template/Primer Preparation 3.3 Sample Preparation 3.4 Performing the Analytical Ultracentrifugation Experiments 3.5 Data Analysis 4 Notes References Chapter 20: Studying RNA-Protein Complexes Using X-Ray Crystallography 1 Introduction 2 Materials 3 Methods 3.1 Tools to Predict the Fold of RNA 3.2 Modeling RNA-Protein Interactions 3.3 Preparing RNase-Free Solutions 3.4 RNA Synthesis 3.5 Determining the RNA Concentration 3.6 Construct Design 3.7 Protein Production 3.8 RNA Binding Assays 3.8.1 EMSA 3.8.2 Biophysical Characterization of RNA-Protein Interactions 3.9 Forming the RNA-Protein Complex 3.10 Setting Up RNA-Protein Crystallization Experiments 3.11 Optimizing Crystallization Conditions 3.12 Crystal Harvesting, Freezing, and Data Collection 3.13 Determining the Structure 3.14 Model Building Using COOT 3.14.1 Building an RNA Chain Using RCrane 3.15 Validating the RNA Structure 3.16 Analyzing the RNA-Protein Structure 3.17 What To Do When It Is Not a Complex 4 Notes References Part VI: Membrane Binding Chapter 21: Flow Linear Dichroism of Protein-Membrane Systems 1 Introduction 2 Materials 3 Methods 3.1 Small Unilamellar Liposome Preparation 3.2 Flow LD Data Collection 3.3 Liposome LD Analysis 3.3.1 Data Processing 3.3.2 Data Interpretation 4 Notes References Chapter 22: Probing Protein-Membrane Interactions and Dynamics Using Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) 1 Introduction 2 Materials 2.1 Instrumentation Buffers 2.2 HDX-MS Setup Buffers 2.3 Description of Mass Spectrometer and Fluidics Setup 3 Methods 3.1 Setting Up the HDX 3.1.1 Experiment Design 3.1.2 Execution of HDX 3.2 Operating the Mass Spectrometer 3.2.1 Precautions Necessary When Starting a Project 3.2.2 Starting Up the MS-LC System Prior to Running Samples 3.2.3 Sample Running 3.3 Data Analysis 3.3.1 MS/MS Analysis Using PEAKS7 3.3.2 Measuring Deuterium Incorporation with HDExaminer 3.3.3 Starting a Project on HDExaminer 3.3.4 Data Processing of Deuterated Samples 3.3.5 Data Analysis and Presentation of Deuterium Incorporation and Differences Between Conditions 4 Notes References Index