Structure and Function of Membrane Proteins (Methods in Molecular Biology, 2302)

✍ Scribed by Ingeborg Schmidt-Krey (editor), James C. Gumbart (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 359
• Category: Library

✦ Synopsis

This book examines detailed experimental and computational approaches for the analysis of many aspects vital to the understanding of membrane protein structure and function. Readers will receive guidance on the selection and use of methods for over-expression and purification, tools to characterize membrane proteins within different phospholipid bilayers, direction on functional studies, and approaches to determine the structures of membrane proteins. Detailed experimental steps for specific membrane proteins with critical notes allow the protocols to be modified to different systems. Written for the highly successful Methods in Molecular Biology series, chapters include the kind of practical information and implementation advice that leads to excellent, reproducible results.
Authoritative and up-to-date, Structure and Function Studies of Membrane Proteins serves as an ideal guide for biologists, biochemists, and biophysicists striving to furtherunderstand these essential proteins and their many biological roles.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Expression and Purification of Human Mitochondrial Intramembrane Protease PARL
1 Introduction
2 Materials
2.1 Protein Expression and Purification Materials, Solutions, and Media
2.2 Media
2.2.1 Growth Media Recipes
2.2.2 Expression Media Recipes
2.3 Buffers and Solutions
3 Methods
3.1 Construction of GFP Fusion Vector and Cloning of PARL
3.2 Transformation of Pichia pastoris
3.2.1 Linearizing Plasmid DNA
3.2.2 Preparing Electrocompetent P. pastoris GS115 Cells
3.2.3 Transformation and Plate Screening
3.3 Large Scale Expression of PARL-GFP
3.4 Cell Lysis and Membrane Fraction Isolation
3.5 Detergent Optimization for PARL-GFP
3.6 IMAC Purification of PARL
4 Notes
References
Chapter 2: Reconstitution of Detergent-Solubilized Membrane Proteins into Proteoliposomes and Nanodiscs for Functional and Str...
1 Introduction
2 Materials
2.1 Preparation of Detergent-Solubilized Phospholipid Stock
2.2 Reconstitution into Proteoliposomes by Dialysis
2.3 Polystyrene Bead Stock Preparation for Proteoliposome and Nanodisc Reconstitution
2.4 Reconstitution into Proteoliposomes via Polystyrene Beads
2.5 Small-Volume Reconstitution (100 μL or Less) into Nanodiscs
2.6 Large-Volume (1-3 mL) Reconstitution into Nanodiscs
3 Methods
3.1 Preparation of Detergent-Solubilized Phospholipid Stock
3.2 Preparation of Polystyrene Beads
3.3 Reconstitution into Proteoliposomes by Dialysis
3.4 Reconstitution into Proteoliposomes via Polystyrene Beads
3.5 Reconstitution into Nanodiscs
3.5.1 Small-Volume Reconstitution (100 μL or Less) into Nanodiscs
3.5.2 Large-Volume (1-3 mL) Reconstitution into Nanodiscs
4 Notes
References
Chapter 3: Biochemical Characterization of GPCR-G Protein Complex Formation
1 Introduction
2 Materials
2.1 Protein Samples and Chemicals
2.2 Affinity Purification
2.3 Gel Filtration
3 Methods
3.1 Solubilization
3.2 Affinity Purification of Opsin
3.3 Reconstitute Opsin into Inactive and Active Rhodopsin
3.4 Form G Protein Heterotrimer Gαi/Gβγt
3.5 Prepare Gel Filtration Control Samples
3.6 Prepare Rhodopsin-G Protein Complex Samples for Gel Filtration
3.7 Prepare Rhodopsin-G Protein Complex Dissociation Sample for Gel Filtration
3.8 Gel Filtration
4 Notes
References
Chapter 4: Electrophysiological Approaches for the Study of Ion Channel Function
1 Introduction
2 Materials
2.1 Solutions
2.2 Reagents
2.3 Hardware and Software
3 Methods
3.1 Recording from Channels Heterologously Expressed in Xenopus Oocytes
3.1.1 Preparation of Xenopus Oocytes and CFTR cRNAs
3.1.2 Inside-Out Single Channel Recording
3.1.3 Inside-Out Macropatch Recording
3.1.4 Two-Electrode Voltage-Clamp Recording
3.2 Recording Channel Currents in Epithelial Cell Monolayers Using the Ussing Chamber
3.2.1 Running an Ussing Chamber Experiment
3.3 Recording from Channels Reconstituted into Planar Lipid Bilayers
3.3.1 Setting Up the Equipment for a Planar Lipid Bilayer Experiment
3.3.2 Preparing for a Planar Lipid Bilayer Experiment
3.3.3 Running a Planar Lipid Bilayer Experiment
References
Chapter 5: Isothermal Titration Calorimetry of Membrane Proteins
1 Introduction
2 Materials
2.1 Buffer Preparation
2.2 Bicelle Preparation
2.3 Preparation of Integrin αIIb and β3TM Peptides
3 Methods
3.1 Preparation of Calorimeter
3.2 Loading of Sample Cell
3.3 Loading of Injection Syringe
3.4 Setting of ITC Run Parameters
3.5 Subtraction of Heat of Ligand Dilution and Data Fitting
3.6 Conversion of KPL and DeltaG to the Mole Fraction Scale
4 Notes
References
Chapter 6: Atomic Force Microscopy Reveals Membrane Protein Activity at the Single Molecule Level
1 Introduction
2 Materials
3 Methods
3.1 Choosing the Supporting Surface
3.1.1 Subsurface Preparation
3.2 Mica Preparation
3.3 Glass Preparation
3.3.1 Wet Etching of the Glass
3.3.2 Mounting the Glass
3.3.3 Final Glass Cleaning
3.4 Reconstitution of Purified Membrane Protein System
3.4.1 To Form Liposomes
3.4.2 To Form Proteoliposomes
3.5 Biochemical Activity Assay of Surface-Adsorbed Translocase Complexes
3.5.1 ATP Hydrolysis Assay
3.5.2 Protein Translocation Activity Assay
3.6 AFM Imaging of Membrane Proteins
3.6.1 Buffer Considerations
3.6.2 Preparing the Sample
3.7 Imaging
3.7.1 Force Control and Feedback Tuning
3.7.2 Large Scale Imaging
3.7.3 Kymographs
3.8 Analysis of AFM Images of Membrane Proteins
3.8.1 Hessian Blob Algorithm
3.8.2 Tracking Individual Proteins Over Time
3.8.3 Precision Kymograph Analysis via the Line Detection Algorithm
4 Notes
References
Chapter 7: Structure Determination of Membrane Proteins Using X-Ray Crystallography
1 Introduction
2 Materials
2.1 Cloning
2.1.1 Polymerase Chain Reaction (PCR)
2.1.2 Agarose Gel Electrophoresis
2.1.3 DNA Gel Extraction/PCR Cleanup
2.1.4 Restriction Enzyme Digestion
2.1.5 Ligation of Insert and Vector
2.1.6 Transformation of Ligation Reaction into Chemically Competent DH5α
2.1.7 Overnight Cultures
2.1.8 DNA Plasmid Minipreps
2.1.9 DNA Sequencing Analysis
2.2 Expression
2.2.1 Transformation of Clone into BL21(DE3) Cells
2.2.2 Small-Scale Expression Test
2.2.3 Prepare a Glycerol Stock of the Transformed Cells
2.2.4 Preparing Cells for SDS-PAGE Analysis
2.2.5 Prepare a 5 mL Overnight Starter Culture
2.2.6 Large-Scale Expression
2.3 Purification
2.3.1 Preparation of Lysate
2.3.2 Solubilization from Whole Cell Lysate
2.3.3 Solubilization from Isolated Membranes
2.3.4 Isolation of Inclusion Bodies and Refolding
2.3.5 Purification of Solubilized Membrane Proteins
2.4 Crystallization
2.4.1 Aliquoting Commercial Crystallization Broad-Matrix Screens (Hanging Drop)
2.4.2 Preparing 96-Well Optimization Screens
2.4.3 Performing Broad-Matrix and Optimization Crystallization Screening Using an Automated Crystallization Robot (Hanging and...
2.4.4 Performing Optimization Screening Manually
2.4.5 Visualizing and Assessing Crystallization Leads
2.5 Data Collection and Structure Determination
2.5.1 Harvesting Crystals
2.5.2 Storing Crystals
2.5.3 Screening and Data Collection
2.5.4 Diffraction Analysis and Structure Determination
3 Methods
3.1 Cloning
3.1.1 Polymerase Chain Reaction (PCR)
3.1.2 Agarose Gel Electrophoresis and DNA Gel Extraction/PCR Cleanup
3.1.3 Restriction Enzyme Digestion
3.1.4 Ligation of Insert and Vector
3.1.5 Transformation of Ligation Reaction into Chemically Competent DH5α
3.1.6 Overnight Cultures and DNA Plasmid Minipreps
3.1.7 DNA Sequencing Analysis
3.2 Expression
3.2.1 Transformation into BL21 (DE3) Cells
3.2.2 Small-Scale Expression Test
3.2.3 Preparing Cells for SDS-PAGE Analysis
3.2.4 Prepare a Glycerol Stock of the Transformed Cells
3.2.5 Prepare a 5 mL Overnight Starter Culture for Large-Scale Expression
3.2.6 Large-Scale Expression
3.3 Purification
3.3.1 Preparation of Lysate
3.3.2 Solubilization from Whole Cell Lysate
3.3.3 Solubilization from Isolated Membranes
3.3.4 Isolation of Inclusion Bodies and Refolding
3.3.5 Purification of Solubilized Membrane Proteins
3.4 Crystallization
3.4.1 Aliquoting Commercial Crystallization Broad-Matrix Screens (Hanging Drop)
3.4.2 Preparing 96-Well Optimization Screens
3.4.3 Performing Broad-Matrix and Optimization Crystallization Screening Using an Automated Crystallization Robot (Hanging and...
3.4.4 Performing Optimization Screening Manually
3.4.5 Visualizing and Assessing Crystallization Leads
3.5 Data Collection and Structure Determination
3.5.1 Harvesting and Storing Crystals
3.5.2 Screening and Data Collection
3.5.3 Diffraction Analysis and Structure Determination
4 Notes
References
Chapter 8: Studying Membrane Protein Structures by MicroED
1 Introduction
2 Materials
3 Methods
3.1 Preparing the Grids
3.2 Blotting
3.2.1 Blotting Conditions for the Standard MicroED Workflow
3.2.2 Blotting Conditions for the CryoFIB Workflow
3.3 (Optional) Transfer to FIB/SEM
3.4 (Optional) FIB Milling Crystals
3.5 Transfer Grids into the TEM
3.6 Collection of MicroED Data from Crystals
4 Notes
References
Chapter 9: Single-Particle Cryo-EM of Membrane Proteins
1 Introduction
1.1 Sample Optimization in Negative-Stain EM
1.2 Cryo-EM Single-Particle Analysis
2 Materials
2.1 Negative-Stain EM
2.2 Cryo-EM
2.3 Single-Particle Analysis
3 Methods
3.1 Sample Preparation for Cryo- and Negative-Stain EM
3.2 Testing Sample Quality in Negative-Stain EM
3.3 Sample Vitrification, Using a Vitrobot
3.4 Screening of the Cryo-EM Grids
3.5 Cryo-EM Data Collection
3.6 Data Processing
4 Notes
References
Chapter 10: Helical Membrane Protein Crystallization in the New Era of Electron Cryo-Microscopy
1 Introduction
2 Materials
2.1 Co-reconstitution of SERCA and Phospholamban
2.1.1 Stock Solutions
2.1.2 Co-reconstitution
2.2 Crystallization
2.3 Electron Cryo-Microscopy
3 Methods
3.1 Reconstitution
3.1.1 Preparation of Reagents for Reconstitution
3.1.2 Preparation of Lipid-Peptide Thin-Films
3.1.3 Coreconstitution of SERCA/Peptide into Proteoliposomes
3.2 Crystallization
3.2.1 Preparation of 2D Crystals
3.3 Cryo-EM
3.3.1 Sample Preparation for Cryo-EM Negative Stain Screen
3.3.2 Electron Cryo-Microscopy
3.3.3 Image Processing Scheme
4 Notes
References
Chapter 11: NMR Spectroscopic Studies of Ion Channels in Lipid Bilayers: Sample Preparation Strategies Exemplified by the Volt...
1 Introduction
2 Materials
2.1 Reagents
2.2 Media Recipes
2.3 Buffers
3 Methods
3.1 Overexpression of hVDAC1 in E. coli
3.2 Isolation of Inclusion Bodies
3.3 Purification of hVDAC1 from Inclusion Bodies
3.4 Refolding of hVDAC1 into Detergent Micelles
3.5 Purification of Natively Folded hVDAC1 in Detergent Micelles
3.6 Formation of hVDAC1 2D Lipid Crystals
4 Notes
References
Chapter 12: Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering
1 Introduction
2 Materials
2.1 Molecular Biology
2.2 Transformation of a Suitable E. coli Host
2.3 Media and Solutions
2.4 Growth of Deuterated Inoculum
2.5 Preparation of the Bioreactor Vessel and Accessories
2.6 Expression of Deuterated Membrane Protein
2.7 Membrane Isolation from Harvested Cell Paste
2.8 Purification of Deuterated Membrane Protein
3 Methods
3.1 Molecular Biology
3.2 Transformation of a Suitable E. coli Host
3.3 Preparation of Media and Solutions
3.4 Growth of the Deuterated Inoculum
3.5 Preparation of the Bioreactor Vessel and Accessories
3.6 Expression of Deuterated Membrane Protein
3.7 Membrane Isolation from Harvested Cell Paste
3.8 Purification of Deuterated Membrane Protein
4 Notes
References
Chapter 13: Preparing Membrane Proteins for Simulation Using CHARMM-GUI
1 Introduction
2 Materials
2.1 Programs
2.2 Hardware
3 Methods
3.1 Read Protein Coordinates and Manipulate Structure
3.1.1 Load PDB File
3.1.2 Manipulate PDB File
3.2 Orient the Protein
3.2.1 Orient and Position Protein
3.2.2 Generate Pore Water
3.3 Determine the System Size
3.4 Build the Components
3.5 Assemble the Components
4 Notes
References
Chapter 14: Coarse-Grained Molecular Dynamics Simulations of Membrane Proteins: A Practical Guide
1 Introduction
2 Materials
2.1 Obtaining the Protein Structure
2.2 Software
2.3 Hardware
3 Methods
3.1 Conversion to a Coarse Grained Representation
3.2 Embedding the Protein into a Membrane
3.3 Simulation Parameters
3.3.1 Thermostat
3.3.2 Barostat
3.3.3 Nonbonded Interactions
3.3.4 Van der Waals Interactions
3.3.5 Electrostatics Interactions
3.3.6 Polarizable Water and Electrostatics
3.3.7 Time-Step
3.4 Conversion Between Coarse-Grained and Atomistic Representations
4 Notes
References
Chapter 15: Continuous Constant pH Molecular Dynamics Simulations of Transmembrane Proteins
1 Introduction
1.1 pH-Dependent Conformational Dynamics of Membrane Proteins
1.2 Continuous Constant pH Molecular Dynamics Methods
1.3 Membrane-Enabled Hybrid-Solvent CpHMD with pH Replica-Exchange
2 Methods
2.1 Setting Up a CpHMD Simulation for Transmembrane Proteins
2.1.1 Stage 1: Prepare Protein-Bilayer Complex
2.1.2 Stage 2: Equilibrate Bilayer
2.1.3 Stage 3: Perform CpHMD Titration Simulations
2.1.4 Settings in Molecular Dynamics
2.1.5 CpHMD Specific Settings
2.2 pKa Calculations
References
Chapter 16: Molecular Dynamics-Based Thermodynamic and Kinetic Characterization of Membrane Protein Conformational Transitions
1 Introduction
2 Theory
2.1 Introduction
2.2 String Method with Swarms of Trajectories (SMwST)
2.3 Bias Exchange Umbrella Sampling (BEUS)
2.4 Transition Rate Estimation
3 Methods
3.1 Initial Preparation
3.2 Path Generation: Nonequilibrium Pulling Simulations
3.3 Path Optimization: String Method with Swarms of Trajectories
3.4 Free Energy Calculations: Bias-Exchange Umbrella Sampling
3.5 Transition Rate Estimation
4 Notes
References
Chapter 17: Concepts, Practices, and Interactive Tutorial for Allosteric Network Analysis of Molecular Dynamics Simulations
1 Introduction
2 Methodological Background
2.1 Generating Interaction Networks
2.2 Important Graph Theory Terminology
2.3 Network Construction
2.3.1 Distance Based Topology Using a Collision Padding Radius
2.3.2 Correlated Atomic Motion Weights
2.3.3 Energy Based Topology/Weights
2.4 Analyzing Interaction Network Paths
2.4.1 Computing Current Flow Betweenness and Interaction Paths
2.4.2 Computing and Analyzing Node Betweenness
References
Chapter 18: Large-Scale Molecular Dynamics Simulations of Cellular Compartments
1 Introduction
2 Materials
2.1 Input Files
2.1.1 PDB
2.1.2 JS Files
2.1.3 Force Field Parameters
2.1.4 Gridforce Files
2.2 Software
2.2.1 VMD
2.2.2 NAMD
2.2.3 Charm++
2.2.4 APBS
2.2.5 ARBD
2.3 Hardware
2.3.1 Example of Applicable Hardware
2.3.2 Hardware Used for Case Study
3 Methods
3.1 Model Building
3.1.1 Arrange Proteins
3.1.2 Build Membrane and Generate Connectivity File
3.1.3 Create and Crop a Solvation Box
3.1.4 Merge Membrane Water Box Assembly with Proteins
3.2 Equilibration
3.2.1 Water
3.2.2 Protein & Lipids
3.3 Analysis
3.3.1 APBS
3.3.2 ARBD
3.4 Conventional Workflow
3.5 Iterative Workflow Using NAMD-EnTK
4 Case Study
4.1 Arrange Proteins
4.2 Initial Equilibration
4.3 Membrane Equilibration
4.4 Water Equilibration
4.5 Production Run
4.6 APBS Analysis
4.7 ARBD Analysis
References
Index