Membrane Lipids: Methods and Protocols

Description

This detailed book explores examples of current in vitro and in silico techniques that are at the forefront of lipid membrane research today. Beginning with methods and strategies associated with the creation and use of lipid membrane models in various research settings, the volume continues with electrical impedance spectroscopy strategies and methods to identify how ions and proteins interact with model lipid bilayers, guidance on lipid bilayer in silico molecular dynamics modeling, novel techniques to explore lipid bilayer characteristics using neutron scattering, IR spectroscopy, and atomic force microscopy (AFM), as well as unique fluorescence techniques. Written in the highly successful Methods in Molecular Biology series style, chapters include introductions to their respective topics, lists of the necessary materials, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and cutting-edge, Membrane Lipids: Methods and Protocols serves as an ideal guide for researchers seeking to further investigate the often complicated world of lipid membrane biophysics. Preface Contents Contributors Chapter 1: Methods for Forming Giant Unilamellar Fatty Acid Vesicles 1 Introduction 2 Materials 2.1 Lipids 2.1.1 Micelle-Based (pH-Decrease) Method 2.1.2 Paper-Based (pH-Increase) Method 2.2 Chemicals 2.3 Equipment 2.3.1 Micelle-Based (pH-Decrease) Method 2.3.2 Paper-Based (pH-Increase) Method 2.3.3 Vesicle Concentration 3 Methods 3.1 Buffers 3.2 Making GUVs Via Micelles (pH-Decrease Method) 3.2.1 Micelles 3.2.2 Vesicles 3.3 Making GUVs Via Paper-Based Thin Film Hydration (pH-Increase Method) 3.4 Processing Vesicles to Remove Unencapsulated Material 3.4.1 Concentrate Using Centrifugal Filter 3.4.2 Concentrate Using Density Difference 4 Notes References Chapter 2: Preparing Ion Channel Switch Membrane-Based Biosensors 1 Introduction 2 Materials 3 Methods 3.1 Biosensor Preparation 3.1.1 Formation of Tethered Components onto Electrodes 3.2 Preparation of Mobile Phase Lipid Bilayer 3.3 The Direct ICS Biosensor Preparation 3.4 The Competitive ICS Biosensor Preparation 3.5 Sample Preparation 3.5.1 Measurement Procedure 4 Notes References Chapter 3: Langmuir-Schaefer Deposition to Create an Asymmetrical Lipopolysaccharide Sparsely Tethered Lipid Bilayer 1 Introduction 2 Materials 2.1 Electrode Selection 2.2 Cartridge Preparation Kit 2.3 Langmuir-Blodgett Trough 2.4 Solutions 3 Methods 3.1 Preparing Langmuir-Trough 3.2 Langmuir-Shaefer Deposition 3.3 Preparing Cartridges 3.4 Testing the Bilayer Using AC Impedance Spectroscopy 4 Notes References Chapter 4: Electrochemical Impedance Spectroscopy as a Convenient Tool to Characterize Tethered Bilayer Membranes 1 Introduction 2 Materials 2.1 ZView 2.2 Python 2.3 Other 2.4 Solutions 2.5 Chemicals 2.6 Working Solutions 2.7 Proteins (Toxins) 2.8 Equipment 3 Methods 3.1 Sensor Preparation 3.1.1 Surface Cleaning 3.1.2 Magnetron Sputtering 3.1.3 Sensor Surface Functionalization with Thiol SAM 3.1.4 Assembly and Storage of Sensors 3.2 Preparation of Multilamellar Lipid Vesicles (MLVs) 3.3 Vesicle Fusion and Formation of tBLMs 3.4 Electrochemical Impedance Spectroscopy (EIS) Measurements 3.4.1 Setting up EIS Measurements 3.4.2 Testing the Quality of the Anchor SAMs 3.4.3 Real-Time Monitoring of the Vesicle Fusion and tBLM Formation 3.4.4 Post-Vesicle-Fusion Measurements of EIS Spectra of tBLMs 3.4.5 Monitoring and Detection of the Activity of Pore-Forming Toxins 3.5 EIS Data Analysis 3.5.1 EIS Spectra of SAMs 3.5.2 Data Analysis of EIS Spectra of tBLMs 3.5.3 Evaluation of the Natural Defect Densities in Pristine tBLMs 3.5.4 Evaluation of the Densities of Toxin Pores in tBLMs (Homogeneous Approximation) 3.5.5 Evaluation of the Densities of Toxin Pores in tBLMs (Heterogeneous Approximation) 3.5.6 Detection of Defect Clustering in tBLMs 4 Notes References Chapter 5: Measuring Voltage-Current Characteristics of Tethered Bilayer Lipid Membranes to Determine the Electro-Insertion Pr... 1 Introduction 2 Materials 2.1 Electrode Selection 2.2 Cartridge Preparation Kit 2.3 Solutions 3 Methods 3.1 Preparing Cartridges 3.2 Creating a Tethered Bilayer Lipid Membrane Using Solvent Exchange 3.3 Testing the Bilayer Using AC Impedance Spectroscopy 3.4 Measuring the Voltage-Current Characteristics of Tethered Lipid Membranes 3.4.1 Setting up the Potentiostat 3.4.2 The Analysis of the V-I Curve 3.4.3 Voltage Ramps at High Frequency 3.5 The Voltage-Current Characteristics of Tethered Lipid Membrane with Analytes Incorporated in their Structure 4 Notes References Chapter 6: Measuring Activation Energies for Ion Transport Using Tethered Bilayer Lipid Membranes (tBLMs) 1 Introduction 2 Materials 2.1 Membrane Formation 2.2 Temperature Control Setup 2.3 Testing the Bilayer Using AC Impedance Spectroscopy 3 Methods 3.1 Creating a Tethered Bilayer Lipid Membrane (tBLM) Using Solvent Exchange 3.2 Measuring the Conductance-Temperature Dependency of Tethered Lipid Membranes 3.3 Calculating the Activation Energy from the Conductance-Temperature Dependency of Tethered Lipid Membranes 3.4 Conductance-Temperature Measurement Reversibility 4 Notes References Chapter 7: Determining the Pore Size of Multimeric Peptide Ion Channels Using Cation Conductance Measures of Tethered Bilayer ... 1 Introduction 2 Materials 2.1 tBLM Preparation Kit and Software 2.2 Solutions 3 Methods 3.1 Assembling SDx Flow Cell Cartridges 3.2 Creating Tethered Bilayer Lipid Membranes 3.3 Testing of the Lipid Bilayer 3.4 Incorporation of Peptide 3.5 Cation Concentration Gradients 3.6 Data Analysis 3.7 Data Interpretation for Upper Pore Size Limit 3.8 Data Interpretation for Cation Selectivity 4 Notes References Chapter 8: De-Insertion Current Analysis of Pore-Forming Peptides and Proteins Using Gold Electrode-Supported Lipid Bilayer 1 Introduction 2 Materials 2.1 Solutions 2.2 Equipments 2.3 Experimental Setups for Lipid Bilayer Formation and Channel Current Measurements 3 Methods 3.1 PEG-Modified Gold Needle Electrodes 3.2 Measurements of Reconstitution and De-Insertion Currents of Nanopores 3.3 DiCA (De-Insertion Current Analysis) 3.3.1 Step Number Analysis 3.3.2 Pore Conductance Analysis 4 Notes References Chapter 9: Drug Meets Monolayer: Understanding the Interactions of Sterol Drugs with Models of the Lung Surfactant Monolayer U... 1 Introduction 2 Materials: Simulations and Analysis Programs 3 Methods 3.1 Composition of the LSM 3.2 Simulations of the LSM Without Drugs (Reference System) 3.2.1 Set Up of Monolayers at the Vacuum-Water Interface 3.2.2 Energy Minimization and Equilibration of the LSM 3.2.3 Production Runs of the LSM 3.3 Simulations of the LSM with Increasing Drug Concentrations (LSM-Systems) 3.3.1 Drug Concentrations 3.3.2 Setup of LSM-Drug Systems 3.3.3 Energy Minimization and Equilibration of LSM-Drug Systems 3.3.4 Production Runs of the Drug-LSM Systems 3.4 Analysis 3.4.1 Area Per Lipid (APL) 3.4.2 Order Parameter 3.4.3 Lateral Diffusion and MSD of Lipids 3.4.4 Clustering of Drugs 3.4.5 Collapse of Monolayer 4 Notes References Chapter 10: Establishing a Lipid Bilayer for Molecular Dynamics Simulations 1 Introduction 2 Modeling of Lipid Bilayer Structure 2.1 Unix Environment 2.2 Topology and Force Field 3 Computational Details 3.1 Energy Minimization 3.2 Equilibration 3.3 Production MD 3.4 Data Gathering 4 Notes References Chapter 11: Initiating Coarse-Grained MD Simulations for Membrane-Bound Proteins 1 Introduction 2 Materials 2.1 Computer 2.2 Choice of Operating System 2.3 Software 2.3.1 Prerequisites 2.3.2 Coarse-Grained Simulations Linux (Ubuntu) Homebrew (Mac) If git Is Not Installed Linux Mac After git Is Installed 3 Methods: Coarse-Grained Simulations 3.1 Simulation Setup 3.2 Simulation Analysis 4 Notes References Chapter 12: Small-Angle Neutron Scattering of Liposomes: Sample Preparation to Simple Modeling 1 Introduction 2 Materials 2.1 Extruder and Filters 2.2 Small-Angle Neutron Scattering Instruments 3 Methods 3.1 Using an Extruder to Prepare Vesicles 3.2 Small-Angle Neutron Scattering Experiment 3.3 Modelling the Data 4 Notes References Chapter 13: Time-Resolved SANS to Measure Monomer Inter-Bilayer Exchange and Intra-Bilayer Translocation 1 Introduction 2 Materials 2.1 Contrast Match Buffer 3 Methods 3.1 Preparation of Large Unilamellar Vesicles 3.2 TR-SANS Experimentation 3.3 Data Analysis 3.3.1 SANS Data Reduction 4 Notes References Chapter 14: Identifying Membrane Lateral Organization by Contrast-Matched Small Angle Neutron Scattering 1 Introduction 2 Materials 2.1 Small Angle Neutron Scattering Conditions 2.2 Contrast-Matched (CM) Water 3 Methods 3.1 Preparation of Unilamellar Vesicles 3.2 Small Angle Neutron Scattering 3.3 Data Analysis-The Porod Invariant 4 Notes References Chapter 15: Using refnx to Model Neutron Reflectometry Data from Phospholipid Bilayers 1 Introduction 2 Materials 3 Methods 3.1 Solid-Supported Bilayer 3.2 Starting refnx 3.3 Setting Up for Fitting 3.4 Fitting the Data 3.5 Assessing Fit Via Markov Chain Resampling 3.6 MCMC Outputs 4 Notes Appendix References Chapter 16: Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) to Probe Interfacial Water in Floating Bilayer Lipid Me... 1 Introduction 2 Materials 2.1 Cleaning the Prism 2.2 Deposition of Gold Thin Film on Si Prism 2.3 Deposition of fBLM 2.4 SEIRAS Measurement 3 Methods 3.1 Deposition of Gold Film Onto the Planar Surface of Si Hemisphere Prism 3.2 Preparation of Floating Bilayer Lipid Membrane 3.3 Measurement of SEIRA Spectra 4 Notes References Chapter 17: Manipulation of Lipid Membranes with Thermal Stimuli 1 Introduction 2 Materials 2.1 IR Laser-Related Materials and Set-Ups 2.1.1 IR-VIS Set-Up Imaging Module IR Module 2.1.2 Optical Fiber-Associated IR Set-Up 2.2 Solutions 3 Methods 3.1 Preparation of Lipid Samples 3.2 Preparation of an Open Volume Observation (Sample) Chamber 3.3 Application of Thermal Gradients 3.3.1 IR-VIS Through-Objective Set-Up 3.3.2 Optical Fiber-Associated IR Set-Up 3.4 Characterization of Local Temperature 3.4.1 Microthermocouple-Based Measurement 3.4.2 Preparation of a Microthermocouple 3.4.3 Gel-Based Temperature Measurement 4 Notes References Chapter 18: Analyzing Morphological Properties of Early-Stage Toxic Amyloid β Oligomers by Atomic Force Microscopy 1 Introduction 2 Materials 2.1 Amyloid β Solution Preparation 2.2 Amyloid β Sample Preparation for AFM 2.3 AFM Image Processing and Statistical Analysis 3 Methods 3.1 Amyloid β Aliquots Preparation 3.2 Amyloid β Solution Preparation for AFM Measurements 3.3 Amyloid β Sample Preparation for AFM Measurements 3.4 AFM Image Processing and Statistical Analysis 4 Notes References Chapter 19: Formation and Nanoscale Characterization of Asymmetric Supported Lipid Bilayers Containing Raft-Like Domains 1 Introduction 2 Materials 2.1 Solutions 2.2 General Materials 2.3 Equipment 3 Methods 3.1 Preparation of MβCD-SM Complexes 3.1.1 Preparing SM Multilamellar Vesicles (MLVs) 3.1.2 Preparing MβCD Stock Solution 3.1.3 Forming the MβCD-SM Complexes 3.2 DOPC/Chol (3:1 Mole Ratio) SUVs Preparation 3.3 Formation of DOPC/Chol (3:1) SLBs 3.4 Formation of Asymmetric SLBs Through MβCD-Mediated Lipid Exchange 3.5 AFM-Imaging and Force Spectroscopy Measurements 4 Notes References Chapter 20: Rapid FLIM Measurement of Membrane Tension Probe Flipper-TR 1 Introduction 2 Materials 2.1 Labelling Procedure 2.2 TCSPC Instrumentation 2.3 Hypo-osmotic Shock Experiments 3 Methods 3.1 Instrument Set Up 3.2 Image Acquisition 3.3 Analysis and Data Processing 3.4 Analysis 3.5 RapidFLIM of Flipper-TR for Fast Spatio-Temporal Membrane Tension Measurement 4 Notes References Chapter 21: Bacterial Dye Release Measures in Response to Antimicrobial Peptides 1 Introduction 2 Materials 3 Methods 3.1 Bacterial Growth Conditions 3.2 Membrane Potential Sensitive Dye DiSC3(5) 3.3 Nucleic Acid Sytox Green Dye 3.4 Propidium Iodide (PI) and SYTO9 Dye 4 Notes References Chapter 22: Quantitative Measurements of Membrane Lipid Order in Yeast and Fungi 1 Introduction 2 Materials 2.1 Cell Culture and Staining 2.2 Imaging and Analysis 3 Methods 3.1 Sample Preparation 3.2 Mounting Cells into Freshly Prepared Agarose Pad 3.3 Imaging Acquisition 3.4 Image Analysis and Quantification 4 Notes References Index