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Rhodopsin

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Cover of 'Rhodopsin'

Table of Contents

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    Book Overview
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    Chapter 1 The g protein-coupled receptor rhodopsin: a historical perspective.
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    Chapter 2 Rhodopsin Purification from Dark-Adapted Bovine Retina
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    Chapter 3 Mammalian Expression, Purification, and Crystallization of Rhodopsin Variants
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    Chapter 4 Imaging of Rhodopsin Crystals with Two-Photon Microscopy
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    Chapter 5 Functional Stability of Rhodopsin in a Bicelle System: Evaluating G Protein Activation by Rhodopsin in Bicelles
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    Chapter 6 The Rhodopsin-Arrestin-1 Interaction in Bicelles
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    Chapter 7 Detection of structural waters and their role in structural dynamics of rhodopsin activation.
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    Chapter 8 Probing Conformational Changes in Rhodopsin Using Hydrogen-Deuterium Exchange Coupled to Mass Spectrometry
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    Chapter 9 Analysis of Conformational Changes in Rhodopsin by Histidine Hydrogen–Deuterium Exchange
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    Chapter 10 Investigation of Rhodopsin Dynamics in Its Signaling State by Solid-State Deuterium NMR Spectroscopy
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    Chapter 11 Sequential Structural Changes in Rhodopsin Occurring upon Photoactivation
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    Chapter 12 Dynamic Single-Molecule Force Spectroscopy of Rhodopsin in Native Membranes
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    Chapter 13 High-Resolution Atomic Force Microscopy Imaging of Rhodopsin in Rod Outer Segment Disk Membranes
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    Chapter 14 Detection of Rhodopsin Dimerization In Situ by PIE-FCCS, a Time-Resolved Fluorescence Spectroscopy
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    Chapter 15 Oligomeric State of Rhodopsin Within Rhodopsin–Transducin Complex Probed with Succinylated Concanavalin A
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    Chapter 16 Quantification of Arrestin–Rhodopsin Binding Stoichiometry
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    Chapter 17 Rhodopsin Transient Complexes Investigated by Systems Biology Approaches
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    Chapter 18 Rhodopsin
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    Chapter 19 Monitoring of Rhodopsin Trafficking and Mistrafficking in Live Photoreceptors
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    Chapter 20 Measurements of Rhodopsin Diffusion Within Signaling Membrane Microcompartments in Live Photoreceptors
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    Chapter 21 Kinetics of Rhodopsin's Chromophore Monitored in a Single Photoreceptor.
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    Chapter 22 Supplementation with Vitamin A Derivatives to Rescue Vision in Animal Models of Degenerative Retinal Diseases
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    Chapter 23 Sustained Delivery of Retinoids to Prevent Photoreceptor Death
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    Chapter 24 High-Throughput Screening Assays to Identify Small Molecules Preventing Photoreceptor Degeneration Caused by the Rhodopsin P23H Mutation
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    Chapter 25 Gene Therapy to Rescue Retinal Degeneration Caused by Mutations in Rhodopsin
Attention for Chapter 14: Detection of Rhodopsin Dimerization In Situ by PIE-FCCS, a Time-Resolved Fluorescence Spectroscopy
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Chapter title
Detection of Rhodopsin Dimerization In Situ by PIE-FCCS, a Time-Resolved Fluorescence Spectroscopy
Chapter number 14
Book title
Rhodopsin
Published in
Methods in molecular biology, January 2015
DOI 10.1007/978-1-4939-2330-4_14
Pubmed ID
Book ISBNs
978-1-4939-2329-8, 978-1-4939-2330-4
Authors

Adam W. Smith, Smith, Adam W.

Abstract

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 10 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 10 100%

Demographic breakdown

Readers by professional status Count As %
Student > Doctoral Student 2 20%
Student > Bachelor 2 20%
Student > Ph. D. Student 2 20%
Unspecified 1 10%
Student > Master 1 10%
Other 2 20%
Readers by discipline Count As %
Agricultural and Biological Sciences 3 30%
Biochemistry, Genetics and Molecular Biology 3 30%
Unspecified 1 10%
Pharmacology, Toxicology and Pharmaceutical Science 1 10%
Neuroscience 1 10%
Other 1 10%