NMR studies of calcium-bound recoverin containing a polar mvristoyl analog

Recoerin. a new member of the F.F-hand super-family, serves as a Ca" sensor in vision. A myristoyl group eovalentl> attached to the N-termimts of rceovenn enables it to translocate to disc membranes when the t'a" level is elevated. Ca"-bound rccoverin prolongs the lifetime of rhodopsin b blocking its phosphorylation. The NMR solution structure of Ca"'-free recoverin shows that the myristoyl group is sequestered in a deep hvdrophobic cavity (Tanaka el al..;Vr";v 376. 444). The low solubility of Ca -bound myristoylated recorerin has previously prevented determination of its structure. A polar analog of mvristic acid (B-oxa tetradecanoic acid) has been incorporated into recoverin to increase its solubility. 13-oxa rccoverin binds two Ca" with a higher affinity (3 (.tM) than does native recoverin (17 j.iM) and exhibits CV-induced membrane binding. NMR spectra of 3-oa recoverin are similar to spectra of native recoverin. Complete sequencespecific NMR assignments have been obtained for 1 3-oxa recoverin and the NMRdenved structure-4 of Ca"+-bound 13-oxa recoverin is similar to the x-ray erv'stal structure of (Ca" )|-unmyristoylated recoverin and to our recent NMR derived structure of (C a"t):-unmyristoylated form. Four HP-hand motifs are arranged in a linear array, with Ca" -4bound to EF-2 and F.F-3. The structure of the C-ierminal domain is similar in CV-free and (V'-bound recoverin. whereas their Tvterminal domains are different. The amino-terminal myristov I group is sequestered in the C a"'-free protein, whereas the myristoyl group and eight residues from ihc amino terminus are solvent exposed and disordered in the Ca-hound state, liiosvnthetic incorporation of polar analogs of myristic acid mav increase the solubilitv and make feasible the determination of structure of mristo lalcd proteins gêneraiK.