Obtaining and characterization of EF-hand mutants of recoverin

Several EF-hand recoverin mutants mere obtained and their abilities to bind to photoreceptor membranes and to inhibit rhodopsin kinase mere determined. The mutants with the 'spoiled' 2nd, 3rd or (2nd+3rd) EF-hand structures did not act upon the kinase activity in the mu M range of Ca2+ concentrations. Mutations of the 4th EF hand, which 'repaired' its Ca2+-binding activity, resulted in recoverin with three 'working' Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild-type recoverin, containing two working Ca2+-binding structures. (C) 1998 Federation of European Biochemical Societies.