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I.V. Peshenko, A.M. Dizhoor; Ca2+– and Mg2+–Dependent Conformational Changes in Guanylyl Cyclase–Activation Protein 1 (GCAP–1) Revealed by Tryptophan Fluorescence of Its Trp/Phe Mutants . Invest. Ophthalmol. Vis. Sci. 2006;47(13):818.
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GCAPs are Mg2+/Ca2+–binding proteins that activate retinal guanylyl cyclase in the light and inhibit it in the dark as a result of the change in Mg2+ versus Ca2+ binding [1, 2]. The purpose of this study was to identify specific regions in the GCAP–1 molecule that undergo conformational changes as a result of the Mg2+/Ca2+ exchange.
Site directed mutagenesis was used to replace one or two Trp residue of GCAP–1 with Phe. Recombinant bovine GCAP–1 and its Trp/Phe mutants were expressed in E. coli, and their metal–dependent conformational changes were analyzed by measuring tryptophan fluorescence spectra.
We find that transition of GCAP–1 from its metal–free to a Mg2+–bound form is accompanied by decrease in the fluorescence of all three Trp with half–maximal concentration of Mg2+ of 0.14, 0.23 and 0.25 mM for Trp21, Trp51 and Trp94, respectively. Ca2+–dependent changes in the tryptophan fluorescence of GCAP–1 are biphasic. In the absence of Mg2+, binding of Ca2+ results in decrease in the fluorescence of Trp21 and Trp51 with [Ca]1/2 of ∼ 0.015 and 0.026 µM, respectively, and increase in the fluorescence of Trp94 ([Ca]1/2 ∼ 0.12 µM). In the presence of 0.9 mM free Mg2+, binding of Ca2+ results in further decrease in the fluorescence of Trp21 ([Ca]1/2 ∼ 0.12 µM), much less so in case of Trp51. Opposite to Mg2+, binding of Ca2+ increases fluorescence of Trp94 with [Ca]1/2 of ∼ 0.29 µM at 0.9 mM free Mg2+.
At physiological concentration of free Ca2+ and Mg2+ in photoreceptors in the light, all three EF–hands in GCAP–1 are predominantly occupied by Mg2+. Guanylyl cyclase activator form of GCAP–1 is triple Mg2+–bound, with all three Trp residues exposed to the polar environment compared to the metal–free GCAP–1. As free Ca2+ concentration rises in the dark, Ca2+ replaces Mg2+ in all three EF–hands. Binding of Ca2+ in EF–hands 2 and 3 further exposes the Trp21 of the entering α–helix in EF–hand 1 to the solution. Contrary to that, binding of Ca2+ in the EF–hand 4 causes moving of the Trp94 of the entering α–helix of EF–hand 3 back to the non–polar environment. References.  Peshenko & Dizhoor (2004) J. Biol. Chem. 279, 16903–6;  Woodruff et al. (2002) J.Physiol. 542, 843–54.
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