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 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.
Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.|Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Sung-Tae
ST
Yang
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
Sung In
SI
Lim
Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.|Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Volker
V
Kiessling
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.|School of Materials Science and Engineering, and Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea.
Inchan
I
Kwon
Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.|Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Lukas K
LK
Tamm
D000596:Amino Acids / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051603:Recoverin / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000097573:Red Fluorescent Protein
D000596:Amino Acids; D002118:Calcium; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D051603:Recoverin; D000097573:Red Fluorescent Protein