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

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Publication Type Journal Article
School or College School of Medicine
Department Ophthalmology
Creator Baehr, Wolfgang
Other Author Sokal, I; Li, N; Klug, C S; Filipek, S; Hubbell, W L; Palczewski, K
Title Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies
Date 2001
Description 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.
Type Text
Publisher American Society for Biochemistry and Molecular Biology (ASBMB)
Volume 276
Issue 46
First Page 43361
Last Page 43373
Subject Amino Acid Motifs; Cyclic N-Oxides; EF Hand Motifs
Subject MESH Calcium-Binding Proteins; Electron Spin Resonance Spectroscopy
Language eng
Bibliographic Citation Sokal I, Li N, Klug CS, Filipek S, Hubbell WL, Baehr W, Palczewski K. (2001). Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies. J Biol Chem, 276(46), 43361-73
Rights Management (c)American Society for Biochemistry and Molecular Biology (ASBMB)
Format Medium application/pdf
Identifier ir-main,1727
ARK ark:/87278/s6bp0kz3
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2012-06-13
ID 703320
Reference URL https://collections.lib.utah.edu/ark:/87278/s6bp0kz3
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