Purification and Kinetic Analysis of Rhodospirillum Centenum Lov-Kinase and Its Putative Response Regulator

Abstract

LOV histidine kinase (LOV-HK) from the purple photosynthetic bacterium Rhodospirillum centenum was expressed and characterized along with its putative response regulator (RR). LOV-HK and RR were expressed in E. coli and purified in a two-step process of nickel affinity chromatography followed by gel filtration chromatography. Mass spectrometry showed that both proteins were successfully expressed. The LOV-HK dark-state spectrum has broad peaks at 362nm and 450nm characteristic of the flavin mononucleotide (FMN) chromophore in a tight binding pocket. The light-state spectrum showed ~33% bleach at 450nm, which slowly returned to the dark state. The recovery of LOV-HK had a rate constant of 4.13 x 10⁻⁴ S⁻¹ with a half-life of 28.0min. The recovery of LOV-HK was also investigated in the presence of RR and showed a rate of 4.17x 10⁻⁴ S⁻¹ (half-life of 27.7min). These results do not conclusively demonstrate an interaction between the two proteins, however further study is warranted.