Journalartikel

Jahr der Veröffentlichung: 2001

Seiten: 4720-4730

Zeitschrift: European Journal of Biochemistry

Bandnummer: 268

Heftnummer: 17

ISSN: 0014-2956

Open Access Status: Bronze

DOI Link: https://doi.org/10.1046/j.1432-1327.2001.02395.x

Verlag: Wiley: No OnlineOpen

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 approximate to 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 approximate to 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.

Harvard-Zitierstil: Lamparter, T., Esteban, B. and Hughes, J. (2001) Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 : purification, assembly, and quaternary structure, European Journal of Biochemistry, 268(17), pp. 4720-4730. https://doi.org/10.1046/j.1432-1327.2001.02395.x

APA-Zitierstil: Lamparter, T., Esteban, B., & Hughes, J. (2001). Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 : purification, assembly, and quaternary structure. European Journal of Biochemistry. 268(17), 4720-4730. https://doi.org/10.1046/j.1432-1327.2001.02395.x