TAILIEUCHUNG - Báo cáo khoa học: Probing protein–chromophore interactions in Cph1 phytochrome by mutagenesis

We have investigated mutants of phytochrome Cph1 from the cyanobacter-ium SynechocystisPCC6803 in order to study chromophore–protein inter-actions. Cph1D2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. | ềFEBS Journal Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis Janina Hahn1 Holger M. Strauss1 Frank T. Landgraf2 Hortensia Faus Gimenez2 Gunter Lochnit3 Peter Schmieder1 and Jon Hughes2 1 Forschungsinstitut fur Molekulare Pharmakologie Berlin Germany 2 Pflanzenphysiologie Fachbereich Biologie Chemie Justus-Liebig-Universitat Giessen Germany 3 Biochemisches Institut Fachbereich Medizin Justus-Liebig-Universitat Giessen Germany Keywords biliprotein photoreceptor phytochrome site directed mutagenesis structure-function studies Correspondence J. Hahn Forschungsinstitut fur Molekulare Pharmakologie Robert Rossle Str. 10 D-13125 Berlin Germany Fax 49 30 94793169 Tel 49 30 94793316 E-mail hahn@ Received 28 October 2005 revised 27 January 2006 accepted 3 February 2006 doi We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore-protein interactions. Cph1D2 the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. We generated 12 site-directed mutants of Cph1D2 focusing on conserved residues which might be involved in chromophoreprotein autoassembly and photoconversion. Folding phycocyanobilin-binding and Pr fi Pfr photoconversion were analysed using CD and UV-visible spectroscopy. MALDI-TOF-MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red-shifted photochromic holoprotein. H260Q shows UV-visible properties similar to the wild-type at pH but both Pr and Pfr reversibly bleach at pH indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed folding but the residue is not essential for chromophore-protein autoassembly. In D207A whereas

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