Recoverin is a recently-discovered, 202-residue protein that mediates the Ca2+dependent long-term adaptation to continuous illumination in retinal rod cells by regulating guanylate cyclase activity in response to changes in intracellular [Ca2+]. It is a member of the EF hand family of Ca2+-binding proteins. It is also myristoylated at the amino terminus; the myristoyl moiety appears to be an essential component of the regulatory mechanism. Hence, recoverin is a representative """"""""calcium-myristoyl switch"""""""" protein; other members of this group include visinin, brain neurocalcin, Drosophila frequenin, and calcineurin B. This proposal focuses on structure-function studies of recoverin;
specific aims are: 1. Complete the structure determination of nonmyristoylated recombinant recoverin in a single-Ca2+-bound state; native data have been collected to 2.15 angstroms and model building is in progress. 2-3. Solve the structures of a Ca2+-free """"""""on"""""""" state and a Ca2+-saturated """"""""off"""""""" state. 4. Develop an application of molecular modelling that will allow accurate estimation of side chain packing for various backbone conformations, thereby focusing mutagenesis studies on constructs that are likely to be viable and to give definitive answers to the questions posed. 5. Probe the functional significance of Ca2+ exclusion at EF hands I and IV of recoverin by constructing chimeric proteins in which each Ca2+-binding loop is replaced by both a sequence which should bind Ca2+, and an alternative sequence which excludes Ca2+. 6. Map the effector function of recoverin by designing, constructing and assaying recoverin-neurocalcin or recoverin-frequenin chimeras, and additionally by crystallizing and solving the structures of these chimeras. The results of these experiments should provide a detailed understanding at the molecular level of the mechanism of the calcium-myristoyl switch.
