Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca²⁺‐induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca²⁺‐myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on ¹⁵N‐labeled recoverin to probe main chain conformational dynamics. ¹⁵N NMR relaxation data suggest that Ca²⁺‐free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca²⁺ levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca²⁺‐dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca²⁺ saturated extruded state (R): T ? I ? R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ_exδω < 1). The final step (I ? R) is much slower (τ_exδω > 1). The main chain structure of I is similar in part to the structure of half‐saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ? I may transiently increase the exposure of Ca²⁺‐binding sites to initiate Ca²⁺ binding that drives extrusion of the myristoyl group during I ? R. Proteins 2011; © 2011 Wiley‐Liss, Inc.