Conformational changes in guanylyl cyclase-activating protein 1 (GCAP1) and its tryptophan mutants as a function of calcium concentration

Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca2+-binding protein s belonging to the calmodulin superfamily, Ca2+-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. I n this study, we characterized GCAP1 mutants in which three endogenous none ssential Trp residues were replaced by Phe residues, eliminating intrinsic fluorescence. Subsequently, hydrophobic amino acids adjacent to each of the three functional Ca2+-binding loops were replaced by reporter Trp residues . Using fluorescence spectroscopy and biochemical assays, we found that bin ding of Ca2+ to GCAP1 causes a major conformational change especially in th e region around the EF3-hand motif, This transition of GCAP1 from an activa tor to an inhibitor of Ge requires an activation energy E-a = 9.3 kcal/mol, When Tyr(99) adjacent to the EF3-hand motif was replaced by Cys, a mutatio n linked to autosomal dominant cone dystrophy in humans, Cys(99) is unable to stabilize the inactive GCAP1-Ca2+ complex. Stopped-flow kinetic measurem ents indicated that GCAP1 rapidly loses its bound Ca2+ (k(-1) = 72 s(-1) at 37 degrees C) and was estimated to associate with Ca2+ at a rate (k(1) > 2 x 10(8) M-1 s(-1)) close to the diffusion limit, Thus, GCAP1 displays ther modynamic and kinetic properties that are compatible with its involvement e arly in the phototransduction response.