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Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies. J Biol Chem 2001 Nov 16;276(46):43361-73



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Scopus ID

2-s2.0-0035900764   34 Citations


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.

Author List

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


Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Motifs
Amino Acid Sequence
Calcium-Binding Proteins
Chromatography, Gel
Cyclic N-Oxides
Dose-Response Relationship, Drug
EF Hand Motifs
Electron Spin Resonance Spectroscopy
Electrophoresis, Polyacrylamide Gel
Enzyme Activation
Fluorescent Dyes
Guanylate Cyclase
Guanylate Cyclase-Activating Proteins
Models, Biological
Models, Chemical
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Binding
Protein Conformation
Protein Structure, Tertiary
Sequence Homology, Amino Acid
Spectrometry, Fluorescence
Spin Labels
jenkins-FCD Prod-399 190a069c593fb5498b7fcd942f44b7bc9cdc7ea1