Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.
Sci Rep
; 6: 32866, 2016 09 08.
Article
em En
| MEDLINE
| ID: mdl-27605302
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 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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
/
Membrana Celular
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Recoverina
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Corantes Fluorescentes
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article