Calcium-dependent conformational changes in guanylate cyclase-activating protein 2 monitored by cysteine accessibility

Guanylate cyclase-activating protein 2 (GCAP2) is expressed in vertebrate photoreceptors cells where it regulates the activity of membrane bound guanylate cyclases in a Ca2+-dependent manner. The essential trigger step involves a Ca2+-induced conformational change in GCAP2. We investigated these Ca2+-dependent changes by probing the cysteine accessibility in wild type and mutant GCAP2 forms with the thiol-modifying reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB). Cysteine residues in position 35 and 111 displayed a restricted accessibility in the presence of Ca2+, whereas cysteine in position 131 reacted with DTNB in the presence and absence of Ca2+. Our data indicate that the Ca2+-sensitivity of GCAP2 is significantly controlled by its third Ca2+-binding site, EF-hand 3.