Open AccessSite-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
Author(s) -
Seun-Ah Yang,
Sung In Lim,
Volker Kiessling,
Inchan Kwon,
Lukas K. Tamm
Publication year - 2016
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep32866
Subject(s) - myristoylation , membrane , recoverin , fluorescence , rhodopsin , biophysics , chemistry , biochemistry , green fluorescent protein , bimolecular fluorescence complementation , membrane protein , microbiology and biotechnology , biology , gene , retinal , physics , quantum mechanics
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 Ca 2+ -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.