Ca 2+ ‐dependent conformational changes in the neuronal Ca 2+ ‐sensor recoverin probed by the fluorescent dye Alexa647

Recoverin belongs to the superfamily of EF‐hand Ca 2+ ‐binding proteins and operates as a Ca 2+ ‐sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca 2+ ‐dependent manner. Ca 2+ ‐dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide‐thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca 2+ ‐myristoyl switch of labeled recoverin was monitored by Ca 2+ ‐dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca 2+ ‐concentration was varied, labeled myristoylated recoverin showed a 37%‐change in fluorescence emission and a 34%‐change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time‐resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca 2+ ‐induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant Rec E 85Q that has a disabled EF‐hand 2, but no such influence was detected with the mutant Rec E 121Q (EF‐hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group. Proteins 2007. © 2006 Wiley‐Liss, Inc.