Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies

Guanylyl cyclase-activating proteins are EF-hand Ca2+-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GC AP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions o f the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca2+ binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodo acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environme ntally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline- 3-methyl)methanethiosulfonate, a spin label reagent, were studied using flu orescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a functio n of Ca2+ concentration. No Ca2+-dependent oligomerization of GCAP1 was obs erved at physiologically relevant Ca2+ concentrations, in contrast to the o bservation reported by others for GCAP2. Based on these results and previou s studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca2+ dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motif s and thus switching its partner, guanylyl cyclase, from an inactive (or lo w activity) to an active conformation.