Ca2+– and Mg2+–Dependent Conformational Changes in Guanylyl Cyclase–Activation Protein 1 (GCAP–1) Revealed by Tryptophan Fluorescence of Its Trp/Phe Mutants

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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⁺.

Conclusions: : 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. [1] Peshenko & Dizhoor (2004) J. Biol. Chem. 279, 16903–6; [2] Woodruff et al. (2002) J.Physiol. 542, 843–54.

Keywords: photoreceptors • calcium • protein structure/function