Abstract
Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca2+-binding proteins belonging to the calmodulin superfamily. Ca2+-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. In this study, we characterized GCAP1 mutants in which three endogenous nonessential 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 binding of Ca2+ to GCAP1 causes a major conformational change especially in the region around the EF3-hand motif. This transition of GCAP1 from an activator to an inhibitor of GC requires an activation energy Ea = 9.3 kcal/mol. When Tyr99 adjacent to the EF3-hand motif was replaced by Cys, a mutation linked to autosomal dominant cone dystrophy in humans, Cys99 is unable to stabilize the inactive GCAP1-Ca2+ complex. Stopped-flow kinetic measurements 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 (k1 > 2 x 10(8) M-1 s-1) close to the diffusion limit. Thus, GCAP1 displays thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response.
Highlights
Introduction of Trp into PositionsAdjacent to EF2, EF3, and EF4 in GCAP1(wϪ)—These mutations were generated with a site-directed mutagenesis kit (Qickchange, Stratagene)
We asked what role do the endogenous Trp residues play in the function of GCAP1, in its conformational changes coupled to Ca2ϩ binding and switching from activator to inhibitor of guanylyl cyclase (GC)? Can functional protein variants be produced that provide spectroscopic signals coupled to individual Ca2ϩ-binding sites? If so, what are the contributions of each of these binding sites to the functionally important conformational changes? What are the kinetics of Ca2ϩ binding and release and associated conformational and functional switching? We found that Ca2ϩ binding at the EF3hand motif evokes the largest conformational change, compared with other EF-hand motifs
Trp Residues in Native and Mutant GCAP1—Trp fluorescence of GCAP1 and its mutants was employed to explore conformational changes within these proteins evoked by Ca2ϩ binding
Summary
Introduction of Trp into PositionsAdjacent to EF2, EF3, and EF4 in GCAP1(wϪ)—These mutations were generated with a site-directed mutagenesis kit (Qickchange, Stratagene). For generation of W2GCAP1(wϪ), the two complementary W2s primer (5Ј-TTG AGA CCT GGG ACT TCA) and W2a primer (5Ј-TGA AGT CCC AGG TCT CAA) were used. For generation of W3-GCAP1(wϪ), the complementary primers W3s (5Ј-AAG CTC TGG GAC GTG GAC and W3a and 5Ј-GTC CAC GTC CCA GAG CTT) were used. For W4-GCAP1(wϪ), the primer pair W4s (5Ј-TTC TCC AAG TGG GAC GTCA) and W4a (5Ј-TGA CGT CCC ACT TGG AGA A) was employed. PVL941bGCAP1(wϪ) (10 ng) was used as a template. To clone the mutant vectors, pVL941bGCAP1 and the three mutant vectors in pBluescriptSK (deleted native Trp, and carrying Trp, Trp, and Trp143, respectively) were digested with StuI at 37 °C and SfiI at 50 °C. The linearized pVL941bGCAP1 vector and the mutant fragments were ligated, transformed, purified, and transfected into HighFive insect cells as described above
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
