title: Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure.
abstract: The phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85-kDa protein that acts as a light-regulated histidine kinase seemingly involved in 'two-component' signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo-Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red-shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, the covalent attachment of PCB is blocked by 1 mM iodoacetamide, a cysteine-derivatizing agent. When PCB was incubated with blocked apo-Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long-wavelength-absorbing form represents the protonated, noncovalently bound bilin. Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue-absorbing band upon incubation with apo-Cph1. On the basis of these data we therefore propose a three-step model for phytochrome autoassembly. Size-exclusion chromatography revealed different mobilities for the apoprotein, red-absorbing Cph1-PCB and far-red-absorbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent molecular masses approximately 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1-PCB was further purified by preparative native electrophoresis, the mobility on size-exclusion chromatography was approximately 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.
authors.affiliation: Freie Universitaet Berlin, Pflanzenphysiologie, Germany. lamparte@zedat.fu-berlin.de
authors.forename: T
authors.initials: T
authors.lastname: Lamparter
authors.forename: B
authors.initials: B
authors.lastname: Esteban
authors.forename: J
authors.initials: J
authors.lastname: Hughes
mesh_terms: D001426:Bacterial Proteins; D002850:Chromatography, Gel; D000458:Cyanobacteria / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D020283:Photoreceptors, Microbial; D010834:Phytochrome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020836:Protein Structure, Quaternary
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D010834:Phytochrome; D011494:Protein Kinases; C413623:Cph1 phytochrome protein, bacteria