Biophysics of Membrane Proteins: Methods and Protocols

Preface
Contents
Contributors
Part I: Biochemistry and Functional Analysis
	Chapter 1: Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells
		Abbreviations
		1 Introduction
		2 Materials
			2.1 Reagents and Buffers for Bacterial Cell Culture
			2.2 Reagents for Total Membrane Purification from Bacterial Culture
			2.3 Reagents and Buffers for Western Blot or Dot  Blot
			2.4 Reagents for Detergent Screening
			2.5 Reagents and Buffers for Purification of His-Tagged Proteins by Immobilized Metal Affinity Chromatography (IMAC)
			2.6 Insect Cell Culture and Baculovirus-Infected Insect Cell (BIIC) Preparation
			2.7 Insect Cell Membrane Preparation and Solubilization
			2.8 Purification of Solubilized Membrane Proteins from Insect Cells
			2.9 Lab Apparatus
		3 Membrane Protein Expression in E. coli
			3.1 Small-Scale Optimization of Protein Expression
				3.1.1 Choosing Expression Plasmids
				3.1.2 Host Strain, Media, and Temperature Screen
			3.2 Quick Screening for Expression Conditions
				3.2.1 Small-Scale Screen for Growing Conditions
				3.2.2 Dot Blots
			3.3 Scaling Up MP Production
				3.3.1 Scaling Up Membrane Preparations
				3.3.2 Detergent Screen
				3.3.3 Scaling Up Membrane Protein Purification Via Batch  IMAC
		4 Membrane Protein Expression in Insect Cells
			4.1 Cell Culture
				4.1.1 Initiating Insect Cell Culture from Frozen Stock
				4.1.2 Cell Maintenance and Cell Counting
					Cell Maintenance
					Cell Counting
				4.1.3 Freezing Insect Cells
			4.2 Transfection of Insect Cells
				4.2.1 Recombinant Bacmid DNA Preparation
				4.2.2 Transfection of Insect Cells with Bacmid  DNA
				4.2.3 Virus Titration (Plaque Assay)
				4.2.4 Virus Amplification
				4.2.5 Using the Titerless Infected-Cell Preservation and Scale-Up (TIPS) Method forVirus Storage and Alterative Infection
			4.3 Optimization of Protein Expression and Membrane Purification
				4.3.1 Finding the Right Construct
				4.3.2 Finding the Right Expression Condition: Virus Titration, Expression  Time
				4.3.3 Small-Scale Expression
				4.3.4 Cell Membrane Preparation with Sucrose Cushion
				4.3.5 Detergent Screening
				4.3.6 Large-Scale Membrane Protein Production
			4.4 Purification of Membrane Proteins by Affinity Chromatography
				4.4.1 His-Tagged Membrane Proteins by Immobilized Metal Affinity Chromatography (IMAC)
				4.4.2 Purification of Flag-Tagged Membrane Proteins by Affinity Chromatography
		5 Notes
		References
	Chapter 2: Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Label...
		1 Introduction
		2 Materials
			2.1 Chemicals and  Kits
			2.2 Buffers and Solutions
			2.3 Biological Materials
			2.4 Sample Preparation Materials
			2.5 Instruments
			2.6 Software
		3 Methods
			3.1 Protein Production and Purification
			3.2 Determination of Protein Concentration
			3.3 Labeling of Proteins
			3.4 Assay Optimization
			3.5 Assay Setup
			3.6 Data Analysis
		4 Notes
		References
	Chapter 3: Rationale for the Quantitative Reconstitution of Membrane Proteins into Proteoliposomes
		1 Introduction
		2 Materials
			2.1 Liposome Preparation
			2.2 Liposome Purification
			2.3 Determination of the Phospholipid and Protein Content
		3 Methods
			3.1 Preparation of Negatively Stained Samples
			3.2 Electron Microscopy Observation
			3.3 Preparation of Liposomes
			3.4 Destabilization of the Liposomes: Determination of the Saturating and Solubilizing Concentrations of Detergent
			3.5 Desorption of the Detergent: Formation of the Proteoliposome
			3.6 Purification of the Proteoliposome Populations
			3.7 Determination of the Phospholipid Content
			3.8 Determination of the Protein Content
			3.9 Determination of the Number of Proteins Per Liposome
		4 Notes
		References
	Chapter 4: Functional Characterization of SLC Transporters Using Solid Supported Membranes
		1 Introduction
		2 Materials
			2.1 Sensor Preparation
			2.2 Measurement
		3 Methods
			3.1 Sensor Preparation
			3.2 SURFE2R N1 Initialization
			3.3 Preparations for the Measurement
			3.4 Sensor Quality Control
			3.5 Measurement of EC50 and Inhibitor
			3.6 Measurement of Negative Control
			3.7 Clean-up
			3.8 Data Analysis and Interpretation
				3.8.1 Current Traces and Calculation of EC50
				3.8.2 Inhibition and Negative Control
		4 Notes
		5 Assay Variations for SGLT1, NCX, OCT2, and EAAT3
			5.1 Workflows
			5.2 Buffer Preparation and EC50
			5.3 Inhibitors and IC50 Values
		6 Technical Description of SURFE2R N1 Instrumentation
		References
	Chapter 5: Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membr...
		Abbreviations
		1 Introduction
			1.1 The Mitochondrial ADP/ATP and ATP-Mg/Pi Carriers as Subjects of Study
			1.2 Applications of Thermostability Assays
			1.3 A Note on the Equipment Used for Thermostability Assays with CPM
		2 Materials
			2.1 Strains and Plasmids
			2.2 Expression and Purification Reagents
			2.3 Reagents for Thermostability Assays Using  CPM
			2.4 Equipment
			2.5 Software
		3 Methods
			3.1 Large-Scale Expression of Wild-Type and Mutant AAC in Yeast
			3.2 Small-Scale Expression of Wild-Type and Mutant APC in Yeast
			3.3 Isolation of Yeast Mitochondria
			3.4 Preparation of Lipid for Protein Purification and Addition in Stability Assays
			3.5 Purification of Wild-Type and Mutant AAC and APC for Stability Assays
			3.6 Basic Thermostability Assay
		4 Applications
			4.1 Effects of Detergents, Lipids, and Inhibitors on the Stability of the Mitochondrial ADP/ATP Carrier
			4.2 Effects of Buffer Composition on the Stability of the Mitochondrial ATP-Mg/Pi Carrier
			4.3 Effects of Crystallization Additives on the Stability of the Mitochondrial ATP-Mg/Pi Carrier
		5 Notes
		References
	Chapter 6: Direct Monitoring of GPCR Reconstitution and Ligand-Binding Activity by Plasmon Waveguide Resonance
		1 Introduction
		2 Materials
			2.1 Detergent-Solubilized CCR5 Receptor
			2.2 Preparation of Liposomes (SUVs)
			2.3 Lipid Bilayer Formation on PWR Sensor and CCR5 Reconstitution
			2.4 Cell Membrane Fragment Capture in the Sensor Surface
			2.5 Ligand-Binding Assays
			2.6 PWR Measurements and Data Analysis
		3 Methods
			3.1 Preparation of the Detergent-Solubilized GPCR Sample
			3.2 Formation of the Planar Lipid Membrane or Capture of Cell Membrane Fragments in the Sensor Surface
			3.3 Receptor Reconstitution in the Lipid Membrane
			3.4 Capture of Cell Membrane Fragments in the Sensor Surface
			3.5 Protein Ligand-Binding Activity
			3.6 PWR Data Analysis
		4 Notes
		5 Summary
		References
Part II: Experimental and Theoretical Structural Determination
	Chapter 7: Examining Membrane Proteins by Neutron Scattering
		1 Introduction
			1.1 Membrane Proteins in Solution
			1.2 SANS for Structural Biology: Specific Parts of Complexes May Be Masked
			1.3 Strategies Developed for Matching Out the Nonmembrane Protein Contribution in SANS
		2 Theoretical Background
			2.1 Scattering Length Density (SLD) and Contrast in SANS
			2.2 Incoherent Scattering: Absorption
			2.3 General Principles of Neutron Scattering
			2.4 Analysis of Neutron Scattering Data
				2.4.1 Analytical Analysis of Neutron Scattering Data
				2.4.2 Modelling the SANS Curves
		3 Instrument Description
			3.1 The Beam Line
			3.2 Samples and Bio-specific Sample Environment on the Beam Line
		4 Prior to the Experiment
			4.1 Defining the Labelling Strategy and Samples for an Optimal Signal on the Beam Line
				4.1.1 Calculating the SLD of the Different Components
				4.1.2 Defining the Labelling Strategy and Samples
			4.2 Designing the Membrane Purification Strategy
				4.2.1 MP and Lipid Deuteration: If Needed
				4.2.2 Detergent and Buffer Exchange Steps
				4.2.3 Reference Buffer
				4.2.4 Control of Protein Homogeneity
			4.3 Defining the Optimal SANS Geometry
			4.4 Application to a Beam Line
		5 Materials
			5.1 The Beam Line
			5.2 To Be Brought to the Beamline
			5.3 Equipment
				5.3.1 Equipment for Solvent Exchange and Sample Concentration
				5.3.2 Equipment for Sample Concentration Measurement
				5.3.3 Equipment for Quality Control
			5.4 Analysis Software
		6 Methods
			6.1 Experiment
				6.1.1 SANS Experiment
				6.1.2 Data Reduction
				6.1.3 Merging, Buffer Subtraction, and Normalization by Protein Concentration
				6.1.4 Determination of the Contrast Match Point
			6.2 Data Analysis
		7 Notes
		Appendix 1: Uncertainty on (I0/c) and Match Point
		References
	Chapter 8: Solution X-Ray Scattering for Membrane Proteins
		1 Introduction
			1.1 Why SAXS for Membrane Proteins
			1.2 Why Use SEC
			1.3 Why Use RI and MALLS
			1.4 Challenge: Modeling of the Data
		2 Outline
		3 General Notes About Sample Preparation and System Operation
			3.1 Thinking in C-CMC
		4 Experimental Procedure
			4.1 Equilibrate the System with the Buffer
			4.2 Sample Preparation and Data Collection
			4.3 Data Processing
		5 Cross-check: Calculation of Number of Detergent Molecules Using Different Methods
		6 Modeling Transmembrane Protein Corona with Memprot: Preparation
			6.1 Aligning the Protein with Rotate_Protein Utility Program (Only for Symmetric Multimeric Proteins)
			6.2 Aligning the Protein with Pymol
			6.3 Memprot Input
		7 Modeling Transmembrane Protein Corona with Memprot: Calculations
		8 Modeling with Dadimodo
		9 Appendix 1: How to Measure dn/dc of a Detergent
		10 Appendix 2
		References
	Chapter 9: Interpreting SAXS/WAXS Data with Explicit-Solvent Simulations: A Practical Guide
		1 Introduction
			1.1 Hands On: Getting Ready
		2 Hands On, Part 1: Calculating SWAXS Curves from MD Trajectories
			2.1 Preparation of the Pure-Solvent System
			2.2 Calculating the SAXS Curve from an .xtc  File
			2.3 Generating the Envelope
			2.4 The SWAXS Calculation
			2.5 Analysis of the Results
		3 Hands On, Part 2: SAXS-Driven MD Simulation, Refining a Structure Against Experimental  Data
		4 Conclusions and Outlook
		5 Appendix 1: Common Error Messages and Problems
		6 Appendix 2: MD Parameters for SAXS Curve Prediction Calculations
		7 Appendix 3: SWAXS Part of a SAXS-Driven MD Simulation
		References
	Chapter 10: Determining the Free Energies of Outer Membrane Proteins in Lipid Bilayers
		1 Introduction
		2 Materials
			2.1 Reagents for Refolding and Unfolding Experiments
			2.2 Equipment
		3 Methods
			3.1 Fluorescence Parameters and Their Usage
			3.2 Preparation of LUVs
			3.3 Preparation of OMP Stock
			3.4 Titration Protocols
				3.4.1 Urea Method
					Preparation of the Unfolded PagP Stock
					Preparation of the Unfolding Titration
					Preparation of the Refolding Titration
					Preparation of Buffer Blanks
					Acquisition and Analysis of Trp-Fluorescence Spectra
				3.4.2 Guanidinium-HCl Method
					Preparation of the Unfolded PagP Stock
					Preparation of the Unfolding Titration
					Preparation of the Refolding Titration
					Acquisition and Analysis of Trp-Fluorescence Data
				3.4.3 Data Fitting of PagP Titrations
				3.4.4 Interpretation of the Measured DeltaG0
			3.5 When Titration Curves Do Not Overlap
				3.5.1 Recognizing Hysteresis
				3.5.2 Troubleshooting Hysteresis
					Screening for Refolding Conditions
					Determining the Nature of the End States
					Aggregation and Competing Folding Pathways
			3.6 Concluding Remarks
		References
	Chapter 11: Interrogating Membrane Protein Structure and Lipid Interactions by Native Mass Spectrometry
		1 Introduction
		2 Native Mass Spectrometry-Based Structural Biology
			2.1 Ionization and Charge State Distributions
			2.2 Collision-Induced Dissociation
			2.3 Ion Mobility Mass Spectrometry
		3 Native Mass Spectrometry of Integral Membrane Proteins
			3.1 The Native Mass Spectrum of  IMPs
			3.2 Instrumentation for Native MS of Membrane Proteins
				3.2.1 Q-ToF-Based Instruments for Native  MS
				3.2.2 Orbitrap Instruments for Native  MS
		4 Buffers and IMP Reconstitution for Native  MS
			4.1 The ``Right´´ Hydrophobic Environment: Crucial for IMPs to Adopt Their Conformationally Defined States
				4.1.1 Detergent-Based Reconstitution Systems
				4.1.2 Detergent-Free Reconstitution Systems
		5 Case Studies
			5.1 Using Ion Mobility to Investigate Conformational Changes
			5.2 Top-Down Sequence Information of Membrane Proteins
			5.3 The Role of Lipids in Membrane Protein Stability and Functionality
			5.4 Native MS Can Assist with High-Resolution IMP Structures
		6 Future Perspectives
		7 Notes
		References
	Chapter 12: Determination of the Molecular Mass of Membrane Proteins Using Size-Exclusion Chromatography with Multiangle Laser...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Equilibration and Calibration of the Detectors
			3.2 Setting Up a Method
			3.3 Running Your Experiment and Calculation of Molar  Mass
			3.4 Cleaning the System
		4 Notes
		References
Part III: Membrane Protein Dynamics and Conformations
	Chapter 13: Dynamics of Membrane Proteins Monitored by Single-Molecule Fluorescence Across Multiple Timescales
		1 Introduction
		2 Materials
			2.1 Expression of SecYEG
			2.2 Purification of SecYEG
			2.3 Labeling of SecYEG
			2.4 Vesicle Preparation and SecYEG Reconstitution into Liposomes
			2.5 Glass Coverslip Preparation and PL Immobilization for TIRF Microscopy
			2.6 GODCAT Photoprotection System for TIRF Imaging
		3 Methods
			3.1 Expression of SecYEG
			3.2 Purification of SecYEG
			3.3 Labeling of SecYEG
			3.4 Preparation of Proteoliposomes Containing Single Labeled SecYEG
			3.5 Coverslip Surface Preparation and Derivatization with  PLs
			3.6 Surface Immobilization of Proteoliposomes
			3.7 TIRF Imaging (100 ms to 100 s Timescale)
			3.8 Confocal Microscope ALEX Data Collection (10 μs to 100 ms Timescale)
			3.9 Nanosecond Time-Resolved FRET Setup and Data Collection
			3.10 Analysis of TIRF-FRET Time Trajectories of Immobilized Samples
			3.11 Data Analysis from Freely Diffusing Samples-Confocal Microscopy
			3.12 Analysis of Dynamics on Millisecond to Microsecond Timescale
			3.13 Analysis of Dynamics on Nanosecond Timescale
		4 Notes
		References
	Chapter 14: Short-Range Distance Measurement by Transition Metal Ion FRET
		1 Introduction
			1.1 tmFRET for Determining Intramolecular Distances Between Protein Domains
			1.2 Monitoring Conformational Dynamics Induced by Manipulation of the Protein
			1.3 Measuring Stability of Protein Secondary Structure
		2 Materials
			2.1 Protein Purification and Fluorescent Labeling
			2.2 Sample Preparation and Data Acquisition
		3 Methods
			3.1 Site-Directed Fluorescent Labeling of Detergent-Solubilized Membrane Protein
			3.2 Sample Preparation
			3.3 Data Acquisition
			3.4 Data Analysis
			3.5 High-Throughput Setup
		4 Notes
		References
	Chapter 15: PELDOR/DEER: An Electron Paramagnetic Resonance Method to Study Membrane Proteins in Lipid Bilayers
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Buffers
			2.3 Equipment
			2.4 Software
		3 Methods
			3.1 Expression of McjD
			3.2 Extraction and Purification of McjD
			3.3 Site-Directed Spin Labeling of McjD for EPR Spectroscopy
			3.4 cw-EPR Spectroscopy
			3.5 Reconstituting McjD into Bicelles
			3.6 Preparation of the PELDOR Sample
			3.7 The PELDOR Experiment
			3.8 PELDOR Data Processing
			3.9 Data Interpretation and Structural Analysis
		4 Notes
		References
Index