| Autor: |
Hahn, Janina, Strauss, Holger M., Landgraf, Frank T., Gimenèz, Hortensia Faus, Lochnit, Günter, Schmieder, Peter, Hughes, Jon |
| Předmět: |
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| Zdroj: |
FEBS Journal; Jun2006, Vol. 273 Issue 12, p1415-1429, 15p, 1 Black and White Photograph, 2 Charts, 9 Graphs |
| Abstrakt: |
We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore–protein interactions. Cph1Δ2, 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 Cph1Δ2, focusing on conserved residues which might be involved in chromophore–protein autoassembly and photoconversion. Folding, phycocyanobilin-binding and Pr→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 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, 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 red irradiation of the ground state leads to bleaching of the red Pr band as in the wild-type, a Pfr-like peak does not arise, implicating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV-Vis spectra of both H260Q under alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re-uptake) during Pr→Pfr photoconversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment [Wagner JR, Brunzelle JS, Forest KT & Vierstra RD (2005) Nature 438, 325–331]. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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