Rhodopsin: Methods and Protocols (Methods in Molecular Biology, 2501)

Description

This volume provides readers with the latest information on the advances made in the field of rhodopsins. The chapters in this book cover topics such as new discoveries and developments; new ways to search for rhodopsins; methods to characterize the function and structure of rhodopsins on a molecular level; nano volume high throughput in meso crystallization, and fourth generation x-ray synchrotron sources. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Rhodopsin: Methods and Protocols is a valuable resource for any scientist and researcher interested in learning more about this developing field. Preface Contents Contributors Chapter 1: Microbial Rhodopsins 1 Introduction 1.1 Historical Overview 2 Phylogeny and New Rhodopsins 3 Functions 3.1 Proton Pumps 3.1.1 Archaeal Outward Proton Pumps (Bacteriorhodopsins) 3.1.2 Bacterial Outward Proton Pumps (Proteorhodopsins) 3.1.3 Inward Proton Pumps (Xenorhodopsins, Schizorhodopsins) 3.2 Cation-Conducting Channelrhodopsins (CCRs) 3.2.1 Chlorophyte Cation-Conducting Channelrhodopsins 3.2.2 Cryptophyte Cation-Conducting Channelrhodopsins 3.2.3 Viral Channelrhodopsins Group 1 (VR1, VirChR1s) 3.3 Anion-Conducting Channelrhodopsins (ACRs) 3.3.1 Engineered Anion-Conducting Channelrhodopsins (eACRs) 3.3.2 Natural Anion-Conducting Channelrhodopsins (nACRs) 3.3.3 Intensely Desensitized Anion-Conducting Channelrhodopsins (MerMAIDs) 3.3.4 Prasinophyte and Viral Anion-Conducting Channelrhodopsins (PyACRs and vPyACRs) 3.3.5 Nonalgal Anion-Conducting Channelrhodopsins with Red-Shifted Absorption (RubyACRs) 3.4 Non-proton ion Pumps 3.4.1 Archaeal Anion Pumps (Halorhodopsins, TSA Rhodopsins) 3.4.2 Bacterial Anion Pumps (NTQ Rhodopsins) 3.4.3 Cation Pumps (NaRs) 3.5 Enzymerhodopsins 3.5.1 Histidine Kinase Rhodopsins (HKR) 3.5.2 Rhodopsin Phosphodiesterase (RhoPDE) 3.5.3 Rhodopsin Guanylyl-Cyclase (RhGC) 3.6 Viral Rhodopsins Group 2 3.7 Heliorhodopsins 4 Structures of New Rhodopsins 4.1 Proteorhodopsins 4.2 Channelrhodopsins 4.3 Ion pumps 4.3.1 Sodium Pumps 4.3.2 Anion Pumps Archaeal Anion Pumps Bacterial Anion Pumps 4.3.3 Inward Proton Pumps (Xenorhodopsins) 4.3.4 Viral Rhodopsins 4.3.5 Heliorhodopsins 5 Optogenetic Applications 6 Outlook References Chapter 2: Molecular Biology of Microbial Rhodopsins 1 Introduction 2 Historical Perspectives 3 Isolation and Purification of Microbial Rhodopsins 4 Molecular Biology of Optogenetics 5 Outlook References Chapter 3: Rhodopsin-Based Optogenetics: Basics and Applications 1 Optogenetics 2 Microbial Rhodopsins and Corresponding Optogenetic Technologies 2.1 Basic Properties of Channelrhodopsins and Modifications for Different Applications 2.2 Microbial Rhodopsins and Alternative Optogenetic Approaches 2.2.1 Optogenetic Actuators 2.2.2 Optogenetic Inhibitors 2.2.3 Other Types of Optogenetic Cell Manipulation 2.3 Development of Optogenetic Techniques Synergetic to Rhodopsins 3 Microbial Rhodopsins Diversity 4 Structural Insights into Channelrhodopsins Function 4.1 X-Ray Structures of Cation and Anion Conducting Channelrhodopsins 4.2 Photocycle of CrChR2 5 Exemplary Optogenetic Applications with Biomedical Background 5.1 Optogenetic Memory Retrieval 5.2 Optogenetic Sight and Hearing Restoration 5.2.1 Hearing Restoration 5.2.2 Sight Restoration 5.3 Deep Brain Stimulation Therapy Using Microbial Rhodopsins 6 Conclusions and Outlook References Chapter 4: Searching Metagenomes for New Rhodopsins 1 Introduction to Metagenomics 2 Discovery of Novel Rhodopsins via Different Metagenomic Approaches 3 Novel Rhodopsins by Bioinformatic Searches References Chapter 5: E. coli Expression and Purification of Microbial and Viral Rhodopsins 1 Introduction 2 Materials 2.1 Bacteria Cultivation Materials 2.2 Bacteria Cultivation Equipment 2.3 Protein Purification Solutions 2.4 Protein Purification Equipment 3 Methods 3.1 Preparation of Producer Cells 3.1.1 Design of the Expression Constructs 3.1.2 Synthesis of the Designed Sequences 3.1.3 Insertion of the Synthesized DNA Fragment into Plasmid DNA Vector of Choice 3.1.4 Transformation of E. coli Expression Strains with the Prepared Plasmids 3.2 Small-Scale Test Expression 3.2.1 IPTG as Inducer 3.2.2 Lactose as Inducer 3.2.3 Choice of the Inducer and Its Concentration 3.3 Upscale Expression Culture Volume 3.4 Protein Isolation and Purification 3.4.1 Basic Steps 4 Notes References Chapter 6: Crystallization of Microbial Rhodopsins 1 Introduction 1.1 Structural Studies of Rhodopsins: Bacteriorhodopsin 1.2 Vapor Diffusion Crystallization of Protein-Detergent Complexes 1.3 In Meso Approach 1.4 Crystallization from Bicelles 1.5 Membrane Fusion Method 2 Materials 2.1 Precipitants 2.2 Lipids 2.3 Equipment for LCP Preparation 2.4 Crystallization Tubes and Plates 2.5 Robotic Systems for Crystallization 3 Methods 3.1 Protein Reconstitution in LCP 3.2 High-Throughput Nanovolume Crystallization 3.3 Large-Volume Crystallization 3.3.1 Scaling Up LCP Crystallization for Growing Large Crystals 3.3.2 Large-Volume LCP Crystallization for Serial Crystallography 4 Notes References Chapter 7: Crystallographic Studies of Rhodopsins: Structure and Dynamics 1 Introduction 2 Minimizing Effects of Radiation Damage 3 Structures of Light-Induced Reaction Intermediates 3.1 General Considerations 3.2 Steady State Illumination: Pre-illumination 3.3 Data Collection Strategies for Reaction Intermediates 3.3.1 Trapping 3.3.2 Time-Resolved Crystallography 3.4 Data Analysis for Time-Resolved Crystallographic Studies 4 Notes References Chapter 8: Time-Resolved UV-VIS Spectroscopy of Microbial Rhodopsins 1 Introduction 2 Laser Flash Photolysis Experiments: Protein Kinetics from Nanoseconds to Seconds 2.1 Optical Layout of the Experimental System and Major Electronic Components 2.2 Data Acquisition 2.3 Global Multi-exponential Nonlinear Least-Square Fitting of the Data 3 Photocycle Modeling 3.1 Model Assumptions: Irreversible Sequential Chain of Reaction Steps with Kinetically Distinct Rate Constants 3.2 Determination of UV-VIS Absorption Spectra of the Transient States (Intermediates) 4 Concluding Remarks References Chapter 9: Solid-State NMR Spectroscopy on Microbial Rhodopsins 1 Introduction 1.1 General Background 1.2 Solid-State NMR Spectroscopy 2 General Sample Preparation Requirements 3 Chromophore and Photocycle Intermediate States 3.1 Light-Induced Cryo-Trapping and NMR Spectroscopy 3.2 The Retinal-Schiff Base Complex During the Photocycle 3.3 Probing-Specific Residues During the Photocycle 4 3D Structure Determination of Microbial Rhodopsins 4.1 Resonance Assignment 4.2 Distance Restraints 4.3 Structure Calculation 4.4 Differences Between the NMR and X-Ray Structures of ASR 4.5 Probing the Conformational Dynamics of Microbial Rhodopsins by Solid-State NMR 5 Oligomer Interactions 5.1 NMR on Mixed-Labeled Oligomers 5.2 Interface Interactions During the Photocycle 5.3 The Use of PREs for Probing Cross-Protomer Interfaces 6 Summary and Perspective References Chapter 10: FTIR and Raman Spectroscopy of Rhodopsins 1 Introduction 2 Materials 3 Methods 3.1 FTIR Spectroscopy 3.1.1 Low-Temperature FTIR Spectroscopy 3.1.2 Time-Resolved FTIR Spectroscopy 3.1.3 Attenuated Total Reflection FTIR Spectroscopy 3.2 Raman Spectroscopy 3.2.1 Resonance Raman Spectroscopy 3.2.2 Time-Resolved Resonance Raman Spectroscopy References Chapter 11: Single-Cell Resolution Optogenetics Via Expression of Soma-Targeted Rhodopsins 1 Introduction: Targeted Optogenetic Molecules 1.1 General Principles: How Do Opsins Work? 1.2 Opsins for Single-Cell Resolution Optogenetics 2 Protocol: Detecting Synaptic Partners Using Single-Cell Resolution Optogenetics in Culture (Fig. 2) 2.1 Materials 2.1.1 Microscopy and Photostimulation 2.1.2 Expressing Single-Cell Opsins 2.1.3 Neural Culture 2.1.4 Electrophysiology 2.2 Methods 2.2.1 Expressing Somatic Opsins in Neural Culture 2.2.2 Preparing the Experimental Setup 2.2.3 Finding the Cells of Interest 2.2.4 Detecting Presynaptic Cells at Single-Cell Resolution Optogenetically 3 Protocol: Detecting Synaptic Partners Using Single-Cell Resolution Optogenetics in Brain Slices (Figs. 3 and 4) 3.1 Materials 3.1.1 Microscopy and Photostimulation Spatial and Intensity Calibration of the Holographic Illumination Characterization of the Holographic Spots Spatial Calibration Intensity Calibration Suppression of the Zero Order of Diffraction 3.1.2 Opsin Expression 3.1.3 Animal Preparation 3.1.4 Electrophysiology 3.2 Methods 3.2.1 Expressing Somatic Opsins in Mouse Visual Cortex: Stereotactic Viral Injections 3.2.2 Acute Brain Slice Preparation 3.2.3 Preparing the Experimental Setup 3.2.4 Detection of Synaptic Connections in Slices with Holographic Photostimulation at Single-Cell Resolution 4 Protocol: Single-Cell Resolution Optogenetics in Mouse Cortex In Vivo (Fig. 5) 4.1 Materials 4.1.1 Microscopy and Photostimulation 4.1.2 Expressing Single-Cell Opsins 4.1.3 Animal Preparation 4.1.4 Electrophysiology 4.2 Methods 4.2.1 Expressing Somatic Opsins in Mouse Visual Cortex 4.2.2 Preparing the Experimental Setup 4.2.3 Holographic Photoactivation of Single or Multiple Cells References Chapter 12: Electrophysiological Characterization of Microbial Rhodopsin Transport Properties: Electrometric and ΔpH Measureme... 1 Introduction 2 Materials 2.1 Proteoliposomes Preparation 2.2 Functional Characterization of Retinal Bearing Proteins on Planar Lipid Bilayers 2.3 Light-Induced Electrogenic Steps of Retinal-Containing Proteins 2.4 Fluorescent Tools for Initial Characterization of Proteoliposome Functionality 3 Methods 3.1 Asolectin Proteoliposomes Preparation 3.2 Functional Characterization of Retinal Bearing Proteins on Planar Lipid Bilayers 3.3 Light-Induced Electrogenic Steps of Retinal-Containing Proteins Measured on Phospholipid-Impregnated Collodion Film 3.3.1 Formation of Phospholipid-Impregnated Collodion Film and Experimental Setup 3.3.2 Electrogenic Steps of bR and ChR2 3.4 Fluorescent Tools for Initial Characterization of Proteoliposome Functionality 3.4.1 Oxonol Fluorescence for the Study of Pumping Activity 3.4.2 Use of 9-Aminoacridine for the Characterization of pH Gradient on Proteoliposomes 4 Notes References Chapter 13: Electrophysiological Characterization of Microbial Rhodopsins by Patch-Clamp Experiments 1 Introduction 2 Materials 2.1 Cell Culture and Transfection 2.2 Electrophysiological Characterization of Microbial Rhodopsins 3 Methods 3.1 Cell Culture and Transfection 3.2 Electrophysiological Characterization of Microbial Rhodopsins 3.3 Intensity Dependence of the Photocurrent 3.4 Wavelength Dependence of the Photocurrent: Action Spectrum 3.5 Investigation of Peak Current Inactivation and Recovery 3.6 Investigation of the Voltage Dependence of the Photocurrent (IV Curve) and Current Density Determination 3.7 Investigation of the Kinetics 3.8 Determination of the Ion Selectivity 4 Notes References Chapter 14: Molecular Optimization of Rhodopsin-Based Tools for Neuroscience Applications 1 Introduction 1.1 Adapting Microbial Genes for Modulation of Neural Function 1.1.1 Open Reading Frame 1.1.2 Untranslated Functional DNA Elements 1.1.3 Protein Trafficking 1.2 Functional Evaluation of Novel Optogenetic Tools In Vitro 1.2.1 Co-expression of Optogenetic Tools with Fluorescent Proteins 1.2.2 Preparation and Transfection of High-Quality DNA for Cultured Cell Transfection 1.2.3 Cell Health and Overexpression 1.3 Optimization of Prokaryotic Rhodopsin Genes for Mammalian Neuron Expression 1.4 Protocol: Preparation of Dissociated Primary Neuronal Cultures 2 Materials 2.1 Materials for Primary Neuron Culture 2.1.1 Neurobasal Media (NB+) 2.1.2 Serum Media (SM) 2.1.3 Hi-Glucose/MEM 2.1.4 Mito+ Serum Extender 2.1.5 5-Fluoro-2′-Deoxyuridine (FUDR) 2.1.6 Dissection Solution 2.1.7 Enzymatic Digestion Solution 2.1.8 Inactivating Solution 2.1.9 Matrigel 2.2 Materials for Preparing Tissue Cultures Plates for Neuron Culture 2.3 Tools for Hippocampal Dissection 2.3.1 Decapitation and Brain Removal: Equipment Needed (Fig. 1) 2.3.2 Hippocampus Dissection: Equipment Needed (Fig. 2) 2.3.3 Workspace: Equipment Needed 2.4 Materials for Tissue Culture Preparations (Fig. 3) 2.4.1 HEK293 Cell Culture Media 2.4.2 Poly-d-Lysine Solution 2.4.3 Tissue Culture Plate Preparation 2.5 Materials for Calcium-Phosphate Transfection of Primary Neuron Cultures 3 Methods 3.1 Preparation of Coverslips and Culture Plates 3.1.1 Preparing Coverslips 3.1.2 Coating of Glass Coverslips with Matrigel 3.2 Tissue Dissection 3.3 Single-Cell Dissociation 3.4 Cell Viability Analysis and Plating 3.5 Glial Inhibition of Dissociated Hippocampal Cultures 3.6 Transfection of Hippocampal Neurons with Optogenetic Constructs 3.7 Calcium Phosphate Transfection 3.8 Preparation of HEK293 Cells for Optogenetic Tool Expression and Testing 3.8.1 Coverslip Preparation 3.8.2 Poly-d-Lysine Coating 3.8.3 HEK Cell Plating References Chapter 15: Optogenetic Studies of Mitochondria 1 Introduction 2 Materials 2.1 Plasmid Construction 2.2 Cell Culture, Plasmid Transfection, and ChR2 Expression 2.3 Optical Illumination and Confocal Imaging (Fig. 1) 2.4 Mitochondrial Membrane Potential Measurement and Cell Death Assay 2.5 Immunocytochemistry 2.6 Mitochondrial Autophagy Assay 3 Methods 3.1 Targeting Express ChR2 in Mitochondrion 3.1.1 Construct pcDNA-ABCB-ChR2-eYFP Plasmid 3.1.2 Determine Mitochondrial ChR2-eYFP Expression by Confocal Microscopy 3.1.3 Confirm Mitochondrial ChR2-YFP Expression in H9C2 Cells by Immunocytochemistry 3.2 Light Illumination Causes Targeted Mitochondrial Depolarization in ABCB-ChR2-eYFP Expressing Cells 3.3 Optogenetic-Mediated Mitochondrial Autophagy (Mitophagy) 3.3.1 Measure Parkin Translocation to Mitochondrion 3.3.2 Analyze Colocalization of Mitochondria and Lysosomes 3.3.3 Measure Mitochondrial Autophagy by Autophagosome Marker LC3 3.4 Optogenetic-Mediated Cell Death 3.4.1 Assess Light Illumination-Mediated Cytotoxicity in ABCB-ChR2-eYFP-Expressing Cells 3.4.2 Determine Time Dependence of Optogenetic-Mediated Cell Death 3.4.3 Determine Light Irradiance Dependence of Optogenetic-Mediated Cell Death 3.4.4 Explore the Mechanistic Pathway (Apoptosis vs. Necrosis) Underlying Optogenetic-Mediated Cell Death 3.5 Optogenetic-Mediated Preconditioning and Cytoprotection 4 Notes References Chapter 16: In Vivo and In Vitro Characterization of Cyclase and Phosphodiesterase Rhodopsins 1 Introduction 2 Materials 2.1 Xenopus Oocytes Preparation and Incubation 2.2 cRNA Preparation and Microinjection 2.3 Fluorescence Check and Storage of Oocytes 2.4 Extraction Buffer and Fluorescence Measurement from Oocytes 2.5 In Vitro Reactions and cAMP/cGMP Immunoassay 2.6 Adjustment of Illumination Conditions 3 Methods 3.1 Preparation of X. laevis Oocytes 3.2 In Vitro Transcription and cRNA Microinjection 3.3 In Vivo Assay with Xenopus Oocytes 3.4 BiFC (Bimolecular Fluorescence Complementation) Experiment 3.5 Extraction of the Crude Membranes from Xenopus Oocytes 3.6 Protein Quantification with YFP Fusion 3.7 In Vitro Reaction of Membrane or Cytosol Extracted from Oocytes 3.8 cGMP and cAMP Measurement 4 Notes References Chapter 17: Optogenetic Control of Human Stem Cell-Derived Neurons 1 Introduction 2 Materials 2.1 In Vitro Expression of Optogenetic Actuators 2.2 Long-Term Neuronal Culture 2.3 Patch Clamp Recording 2.3.1 Extracellular and Intracellular Solutions 2.3.2 Patch Clamp Recordings 2.4 MEA Electrophysiology 2.5 Optogenetic Stimulation Device 3 Methods 3.1 Neuronal Cultures for Patch Clamp Recordings 3.2 Patch Clamp Electrophysiology 3.2.1 Performing Patch Clamp Recordings and Optogenetic Stimulation 3.3 Banker Culture for MEA Electrophysiology 3.4 MEA Electrophysiology 3.4.1 Recording Network Spontaneous Activity 3.4.2 Recording Network Optogenetic-Evoked Activity 4 Notes References Index