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This volume explores the technological improvements in protein engineering, expression, purification, and crystallization of several rhodopsin photoactive intermediates, thus increasing our understanding of rhodopsin activation. The first chapters of the book focus on methods developed to study fundamentals of rhodopsin structure and function, starting with improved purification protocols of native and mutated rhodopsin, followed by methods used for rhodopsin reconstitution into lipid bilayers stabilizing rhodopsin function properties, and finally describing recently developed methods to study structural dynamics of rhodopsin activation and its mechanistic properties. Subsequently, chapters underline various techniques that have been developed to visualize the rhodopsin dimer and to study its functional significance. The next few chapters highlight cutting-edge imaging techniques of photoreceptors, rhodopsin trafficking, and its diffusion within signaling membranes. Finally the book concludes with recent developments that could be potentially beneficial in patient treatments, and treatment strategies for retinal degenerative diseases. Written in the highly successful Methods in Molecular Biology series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory.
Timely and practical, Rhodopsin: Methods and Protocols reaches out to researchers and health practitioners, and provides timely protocol useful for studying structural and functional properties of rhodopsin.
The G Protein-Coupled Receptor Rhodopsin: A Historical Perspective.- Rhodopsin Purification from Dark Adapted Bovine Retina.- Mammalian Expression, Purification, and Crystallization of Rhodopsin Variants.- Imaging of Rhodopsin Crystals with Two-Photon Microscopy.- Functional Stability of Rhodopsin in a Bicelle System: Evaluating G-Protein Activation by Rhodopsin in Bicelles.- The Rhodopsin-Arrestin-1 Interaction in Bicelles.- Detection of Structural Waters and Their Role in Structural Dynamics of Rhodopsin Activation.- Probing Conformational Changes in Rhodopsin Using Hydrogen-Deuterium Exchange Coupled to Mass Spectrometry.- Analysis of Conformational Changes in Rhodopsin by Histidine Hydrogen-Deuterium Exchange.- Investigation of Rhodopsin Dynamics in its Signaling State by Solid-State Deuterium NMR Spectroscopy.- Sequential Structural Changes in Rhodopsin Occurring upon Photoactivation.- Dynamic Single-Molecule Force Spectroscopy of Rhodopsin in Native Membranes.- High-Resolution Atomic Force Microscopy Imaging of Rhodopsin in Rod Outer Segment Disk Membranes.- Detection of Rhodopsin Dimerization in situ by PIE-FCCS, a Time-Resolved Fluorescence Spectroscopy.- Oligomeric State of Rhodopsin within Rhodopsin-Transducin Complex Probed with Succinylated Concanavalin A.- Quantification of Arrestin-Rhodopsin Binding Stoichiometry.- Rhodopsin Transient Complexes Investigated by Systems Biology Approaches.- Three Dimensional Architecture of Murine Rod Cilium Revealed by Cryo-EM.- Monitoring of Rhodopsin Trafficking and Mistrafficking in Live Photoreceptors.- Measurements of Rhodopsin Diffusion within Signaling Membrane Microcompartments in Live Photoreceptors.- Kinetics of Rhodopsin's Chromophore Monitored in a Single Photoreceptor.- Supplementation with Vitamin A Derivatives to RescueVision in Animal Models of Degenerative Retinal Diseases.- Sustained Delivery of Retinoids to Prevent Photoreceptor Death.- High-Throughput Screening Assays to Identify Small-Molecules Preventing Photoreceptor Degeneration Caused by the Rhodopsin P23H Mutation.- Gene Therapy to Rescue Retinal Degeneration Caused by Mutations in Rhodopsin.
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