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Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis.
- Source :
-
The FEBS journal [FEBS J] 2006 Apr; Vol. 273 (7), pp. 1415-29. - Publication Year :
- 2006
-
Abstract
- We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore-protein interactions. Cph1Delta2, 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 Cph1Delta2, 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.
- Subjects :
- Apoproteins chemistry
Apoproteins genetics
Apoproteins metabolism
Bacterial Proteins chemistry
Photoreceptors, Microbial
Phytochrome chemistry
Protein Conformation
Protein Folding
Protein Kinases chemistry
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Bacterial Proteins genetics
Bacterial Proteins metabolism
Mutagenesis, Site-Directed
Phytochrome genetics
Phytochrome metabolism
Protein Kinases genetics
Protein Kinases metabolism
Synechocystis metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1742-464X
- Volume :
- 273
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- The FEBS journal
- Publication Type :
- Academic Journal
- Accession number :
- 16689929
- Full Text :
- https://doi.org/10.1111/j.1742-4658.2006.05164.x