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Originally published In Press as doi:10.1074/jbc.M103614200 on August 27, 2001

J. Biol. Chem., Vol. 276, Issue 46, 43361-43373, November 16, 2001
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Calcium-sensitive Regions of GCAP1 as Observed by Chemical Modifications, Fluorescence, and EPR Spectroscopies*

Izabela SokalDagger , Ning Li§, Candice S. Klug||, SBawomir Filipek**, Wayne L. Hubbell||, Wolfgang Baehr§Dagger Dagger , and Krzysztof PalczewskiDagger Dagger Dagger §§¶¶||||

From the Departments of Dagger  Ophthalmology, §§ Pharmacology, and ¶¶ Chemistry, University of Washington, Seattle, Washington 98195, the § Department of Ophthalmology, Moran Eye Center, University of Utah Health Science Center, Salt Lake City, Utah 84112-5330, the || Jules Stein Eye Institute and the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, and the ** Department of Chemistry, University of Warsaw, 1 Pasteur St, PL-02093 Warsaw, Poland

Guanylyl cyclase-activating proteins are EF-hand Ca2+-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 Ca2+ 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 Cys18, Cys29, Cys106, and Cys125 were observed as a function of Ca2+ concentration. No Ca2+-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca2+ 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 Ca2+ 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.

* This work was supported by National Institutes of Health Grants EY08123 (to W. B.) and EY08061 (to K. P.), a grant from Research to Prevent Blindness, Inc. to the Department of Ophthalmology at the University of Washington and the University of Utah, a Center Grant from Foundation Fighting Blindness, Inc., to the University of Utah, the Ruth and Milton Steinbach Fund, the E. K. Bishop Foundation, and an award from the Alcon Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Novasite Pharmaceuticals, Inc., 3520 Dunhill St., San Diego, CA 92121.

Dagger Dagger Recipients of an Research to Prevent Blindness Senior Investigator Award.

|||| To whom correspondence should be addressed: University of Washington, Dept. of Ophthalmology, Box 356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-221-6784; E-mail: palczews@u.washington.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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