Protein purification of Agrobacterium tumefaciens phytochrome Agp1 and UV-vis spectroscopy analysis in comparison to DrBphP from Deinococcus Radiodurans

Abstract

Phytochromes are bimodal light switchable photosensors exist in plants, bacteria and fungi. Incorporating bilin as their chromophore, most bacteriophytochromes (BphPs) function as two-component histidine kinase affected by red/far-red light and regulate downstream physiological activities in the microorganism. The study aims on the purification of Agrobacterium phytochrome Agp1whose phosphorylation activity in its histidine kinase (HK) domain was recently found to be affected by the temperature. The purified proteins Agp1 were assembled with biliverdin IXα (BV) and monitored by UV-vis spectroscopy with BV incorporated DrBphP from Deinococcus radiodurans. Extinction coefficient spectra of “absolute” Pr and Pfr state of Agp1 together with free BV were drawn. The UV spectra and difference spectra of Agp1 and DrBphP were compared and band assignments were made. According to the evaluation of absorption maximum (λmax) and extinction coefficiency (Ɛ), the near blue region band at 314-320 nm in the difference spectra was assigned to tyrosine absorption. Further analysis of the band assignment and structure of Agp1 and DrBphP reveal that the 314-320 nm band is caused by the interaction of B ring propionate side chain of incorporated chromophore BV with neighboring conserved Tyr216 and/or the D ring carbonyl group with Tyr176 residues. The discovery was evidenced by the literature evaluation of the spectra of tyrosine mutants from BphPs and Synechocystis sp. Cph1. Chromophore and tyrosine interaction as a possible signaling way of BphPs was discussed based on the assignment.