I. Sokal et al., Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies, J BIOL CHEM, 276(46), 2001, pp. 43361-43373
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.