Ca2+‐dependent conformational changes in bovine GCAP‐2

GCAP‐2, a mammalian photoreceptor‐specific protein, is a Ca2+‐dependent regulator of the retinal membrane guanylyl cyclases (Ret‐GCs). Sensing the fall in intracellular free Ca2+ after photo‐excitation, GCAP‐2 stimulates the activity of Ret‐GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP‐2 is a small N‐myristoylated protein containing four EF‐hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP‐2 alters its conformation in response to Ca2+‐binding as measured by a Ca2+‐dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+‐bound and Ca2+‐free forms of GCAP‐2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N‐terminus of Ca2+‐bound GCAP‐2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N‐terminus of GCAP‐2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP‐2 and recoverin, the structural consequences of Ca2+‐binding for these two proteins are significantly dissimilar.