Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies. J Biol Chem 2001 Nov 16;276(46):43361-73
Date
08/29/2001Pubmed ID
11524415Pubmed Central ID
PMC1363678DOI
10.1074/jbc.M103614200Scopus ID
2-s2.0-0035900764 (requires institutional sign-in at Scopus site) 36 CitationsAbstract
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 KAuthor
Candice S. Klug PhD Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid MotifsAmino Acid Sequence
Animals
Calcium
Calcium-Binding Proteins
Cattle
Chromatography, Gel
Cyclic N-Oxides
Cysteine
Dose-Response Relationship, Drug
EF Hand Motifs
Electron Spin Resonance Spectroscopy
Electrophoresis, Polyacrylamide Gel
Enzyme Activation
Eye
Fluorescent Dyes
Guanylate Cyclase
Guanylate Cyclase-Activating Proteins
Mesylates
Models, Biological
Models, Chemical
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Oxadiazoles
Protein Binding
Protein Conformation
Protein Structure, Tertiary
Sequence Homology, Amino Acid
Spectrometry, Fluorescence
Spin Labels
Sulfur