Ca2+-dependent conformational changes in bovine GCAP-2

GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent reg ulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fal l in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the re coverin superfamily, GCAP-2 is a small N-myristoylated protein containing f our EF-hand consensus motifs. In this study, we demonstrate that Like recov erin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-bi nding as measured by a Ca2+-dependent change in its far UV CD spectrum. Dif ferences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP- 2 were also observed by examining their relative susceptibility to V8 prote ase. In contrast to recoverin, we do not observe proteolytic cleavage of th e 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 G CAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity o f GCAP-2 and recoverin, the structural consequences of Ca2+-binding for the se two proteins are significantly dissimilar.