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 Ca(2+)-dependent manner. The essential trigger step involves a Ca(2+)-induced conformational change in GCAP2. We investigated these Ca(2+)-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 Ca(2+), whereas cysteine in position 131 reacted with DTNB in the presence and absence of Ca(2+). Our data indicate that the Ca(2+)-sensitivity of GCAP2 is significantly controlled by its third Ca(2+)-binding site, EF-hand 3.
Helten
Andreas
A
Biochemistry Group, IBU, Faculty V, University of Oldenburg, D-26111 Oldenburg, Germany.
Koch
Karl-Wilhelm
KW
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid; D006162:Guanylate Cyclase / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000378:metabolism
D051604:Guanylate Cyclase-Activating Proteins; D011956:Receptors, Cell Surface; C110715:guanylate cyclase 1; D004228:Dithionitrobenzoic Acid; D006162:Guanylate Cyclase; D003545:Cysteine; D002118:Calcium