Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 - Purification, assembly, and quaternary structure

The phytochrome Cph1 from the cyanobacterium. Synechocystis PCC6803 forms h oloprotein adducts with close spectral similarity to plant phytochromes whe n 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 purifi cation, estimation of the extinction coefficient of holo-Cph1, spectral ana lyses 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 phycoerythrob ilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, t he covalent attachment of PCB is blocked by 1 mM iodoacetamide, a cysteine- derivatizing agent. When PCB was incubated with blocked apo-Cph1, again a s houlder at longer wavelengths appeared. It is therefore proposed that the l ong-wavelength-absorbing form represents the, protonated, noncovalently bou nd 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 mode l for phytochrome autoassembly. Size-exclusion chromatography revealed diff erent mobilities for the apoprotein, red-absorbing Cph1-PCB and far-red-abs orbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent m olecular masses approximate to 200 kDa, a result in agreement with the noti on that autophosphorylation in sensory histidine kinases requires dimerizat ion. When Cph1-PCB was further purified by preparative native electrophores is, the mobility on size-exclusion chromatography was approximate to 100 kD a, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.