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.
Highlights
Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems
The myristoyl chain acts as an anchor for the translocation of cytosolic recoverin to the rod outer segment disk membrane[6,7], a process that is driven by hydrophobic interactions and further enhanced by lysine-mediated electrostatic interactions with the lipid bilayer[8,9]
To minimize perturbation of this important functional switch, potential sites for non-natural amino acid (NNAA) incorporation were examined in the context of physicochemical and functional constraints
Summary
Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. We develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site- 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. To address some of the unresolved issues, we describe here a site-specific labeling procedure that combines the genetic incorporation of a reactive non-natural amino acid with the application of bio-orthogonal chemistry This approach avoids touching the essential Cys[39] and any of the 25 lysines, including at least 5 functionally important lysines near the N-terminus of recoverin that would be randomly modified by amino-reactive labeling techniques[17]. Since many proteins have similar limitations for fluorescent labeling as recoverin, the strategy proposed here will likely be general and prove beneficial for examining a large group of protein-membrane interactions
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