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Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.

ABSTRACT: Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-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 GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of 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 Ca(2+) binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a function of Ca(2+) concentration. No Ca(2+)-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca(2+) concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca(2+) dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.

SUBMITTER: Sokal I

PROVIDER: S-EPMC1363678 | biostudies-literature | 2001 Nov

REPOSITORIES: biostudies-literature

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Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.

Sokal I I Li N N Klug C S CS Filipek S S Hubbell W L WL Baehr W W Palczewski K K

The Journal of biological chemistry 20010827 46

Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-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 GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) an ...[more]

PMID: 11524415

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Project description:Pollen studies are important for the assessment of present and past environment, including biodiversity, sexual reproduction of plants and plant-pollinator interactions, monitoring of aeroallergens, and impact of climate and pollution on wild communities and cultivated crops. Although information on chemical composition of pollen is of importance in all of those research areas, pollen chemistry has been rarely measured due to complex and time-consuming analyses. Vibrational spectroscopies, coupled with multivariate data analysis, have shown great potential for rapid chemical characterization, identification and classification of pollen. This study, comprising 219 species from all principal taxa of seed plants, has demonstrated that high-quality Raman spectra of pollen can be obtained by Fourier transform (FT) Raman spectroscopy. In combination with Fourier transform infrared spectroscopy (FTIR), FT-Raman spectroscopy is obtaining comprehensive information on pollen chemistry. Presence of all the main biochemical constituents of pollen, such as proteins, lipids, carbohydrates, carotenoids and sporopollenins, have been identified and detected in the spectra, and the study shows approaches to measure relative and absolute content of these constituents. The results show that FT-Raman spectroscopy has clear advantage over standard dispersive Raman measurements, in particular for measurement of pollen samples with high pigment content. FT-Raman spectra are strongly biased toward chemical composition of pollen wall constituents, namely sporopollenins and pigments. This makes Raman spectra complementary to FTIR spectra, which over-represent chemical constituents of the grain interior, such as lipids and carbohydrates. The results show a large variability in pollen chemistry for families, genera and even congeneric species, revealing wide range of reproductive strategies, from storage of nutrients to variation in carotenoids and phenylpropanoids. The information on pollen's chemical patterns for major plant taxa should be of outstanding value for various studies in plant biology and ecology, including aerobiology, palaeoecology, forensics, community ecology, plant-pollinator interactions, and climate effects on plants.

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Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.

Publications

Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.

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