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J. Biol. Chem., Vol. 283, Issue 30, 21251-21266, July 25, 2008

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Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion^*

Andrew T. Ulijasz, Gabriel Cornilescu, David von Stetten^¶, Steve Kaminski^¶, Maria Andrea Mroginski^¶, Junrui Zhang, Devaki Bhaya^||1, Peter Hildebrandt^¶, and Richard D. Vierstra²

From the Department of Genetics and National Magnetic Resonance Facility, University of Wisconsin, Madison, Wisconsin 53706, the ^¶Technische Universität, D-10623 Berlin, Germany, and the ^||Department of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B', that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. ¹H-¹⁵N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

Received for publication, February 27, 2008 , and in revised form, April 30, 2008.

^* This work was supported in part by National Science Foundation Grants MCB 0424062 and 07191530 (to R. D. V.), Deutsche Forschungsgemeinschaft Grant SF498 (to P. H. and M. A. M.), and an American Heart Association postdoctoral fellowship (to A. T. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-5.

¹ Supported by the National Science Foundation and the Carnegie Institution of Washington.

² To whom correspondences should be addressed: Dept. of Genetics, 425-G Henry Mall, University of Wisconsin, Madison, WI 53706. Tel.: 608-262-8215; Fax: 608-262-2976; E-mail: vierstra{at}wisc.edu.

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