Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

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 na...

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Main Authors:	Yang, Sung-Tae, Lim, Sung In, Kiessling, Volker, Kwon, Inchan, Tamm, Lukas K.
Format:	Online
Language:	English
Published:	Nature Publishing Group 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015116/

id	pubmed-5015116
recordtype	oai_dc
spelling	pubmed-50151162016-09-12 Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein Yang, Sung-Tae Lim, Sung In Kiessling, Volker Kwon, Inchan Tamm, Lukas K. Article 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. Nature Publishing Group 2016-09-08 /pmc/articles/PMC5015116/ /pubmed/27605302 http://dx.doi.org/10.1038/srep32866 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Yang, Sung-Tae Lim, Sung In Kiessling, Volker Kwon, Inchan Tamm, Lukas K.
spellingShingle	Yang, Sung-Tae Lim, Sung In Kiessling, Volker Kwon, Inchan Tamm, Lukas K. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
author_facet	Yang, Sung-Tae Lim, Sung In Kiessling, Volker Kwon, Inchan Tamm, Lukas K.
author_sort	Yang, Sung-Tae
title	Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
title_short	Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
title_full	Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
title_fullStr	Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
title_full_unstemmed	Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
title_sort	site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
description	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.
publisher	Nature Publishing Group
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015116/
_version_	1613645925627985920

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

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