title: Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis.
abstract: Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca(2+) -induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca(2+)-myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on (15) N-labeled recoverin to probe main chain conformational dynamics. (15) N NMR relaxation data suggest that Ca(2+)-free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca(2+) levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca(2+)-dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca(2+) saturated extruded state (R): T ↔ I ↔ R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ(ex) δω < 1). The final step (I ↔ R) is much slower (τ(ex) δω > 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 Ca(2+)-binding sites to initiate Ca(2+) binding that drives extrusion of the myristoyl group during I ↔ R.
authors.affiliation: Department of Chemistry, University of California, Davis, California 95616, USA.
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Xu
authors.forename: Rieko
authors.initials: R
authors.lastname: Ishima
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D051603:Recoverin / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D009587:Nitrogen Isotopes; D019814:Myristic Acid; D051603:Recoverin; D002118:Calcium