Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
TLDR
Recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events and the site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.Abstract:
Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.read more
Citations
More filters
Journal ArticleDOI
The Binding Properties and Physiological Functions of Recoverin.
TL;DR: This review article focuses on Rcv binding properties to Ca2+, disc membrane and GRK, and its physiological functions in phototransduction and signal transmission.
Journal ArticleDOI
Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry.
Rambabu N. Reddi,Adi Rogel,Efrat Resnick,Ronen Gabizon,Pragati K. Prasad,Neta Gurwicz,Haim Barr,Ziv Shulman,Nir London +8 more
TL;DR: In this article, a covalent ligand directed release (CoLDR) site-specific labeling strategy was proposed, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand.
Journal ArticleDOI
Conductance Changes in Bovine Serum Albumin Caused by Drug-Binding Triggered Structural Transitions.
TL;DR: It is elucidates that protein conductance variations caused by secondary structure transitions are triggered by drug-binding and indicate that electrical methods are of potential application in protein secondary structure analysis.
Journal ArticleDOI
Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein
TL;DR: In this paper, the main focus is on how the fluctuations of free calcium concentrations in photoreceptors between light and dark regulate the activity of retinal membrane guanylyl cyclase (RetGC) via calcium sensor proteins.
Journal ArticleDOI
Zebrafish recoverin isoforms display differences in calcium switch mechanisms
TL;DR: The aims of the present study are to measure specific Ca2-sensing properties of the different recoverin isoforms, to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian recoverin, and to investigate a possible impact of an attached myristoyl moiety.
References
More filters
Journal ArticleDOI
The versatility and universality of calcium signalling
TL;DR: The universality of calcium as an intracellular messenger depends on its enormous versatility, which is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion.
PatentDOI
Synthesis of proteins by native chemical ligation
TL;DR: The technique of native chemical ligation is employable for chemically synthesizing full length proteins as discussed by the authors, which are chemically identical to proteins produced by cell free synthesis, and can be refolded and/or oxidized to form native disulfide-containing protein molecules.
Journal ArticleDOI
Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases
Lee Whitmore,Bonnie A. Wallace +1 more
TL;DR: Recent developments in the analysis of protein secondary structures, including features of the DICHROWEB analysis webserver are discussed, which greatly improve and facilitate the analyses of CD spectra.
Journal ArticleDOI
Fluorescence spectroscopy of single biomolecules.
TL;DR: The progress in applying single-molecule detection and single-Molecule spectroscopy at room temperature by laser-induced fluorescence with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.
Related Papers (5)
Tag-probe labeling methods for live-cell imaging of membrane proteins
Yoshiaki Yano,Katsumi Matsuzaki +1 more