Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis |
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Authors: | Xu Xianzhong Ishima Rieko Ames James B |
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Affiliation: | Department of Chemistry, University of California, Davis, California 95616, USA. |
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Abstract: | 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 (τexδω < 1). The final step (I ? R) is much slower (τexδω > 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. |
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Keywords: | recoverin NMR calcium Ca2+‐myristoyl switch NMR relaxation dispersion analysis vision phototransduction EF‐hand |
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