Structural and Sequence Analyses of an Unusual Bacteriophytochrome from R. palustris

Bacteriophytochromes (Bphs) are bacterial red-light photoreceptors found in photosynthetic and non-photosynthetic bacteria. They undergo reversible photoconversion between two distinct red and far-red light absorbing states, Pr and Pfr, which result in conformational changes within the protein. In order for Pr/Pfr photoconversion to occur, an organic cofactor biliverdin (BV) must be covalently bound to the protein. Bphs are composed of a photosensory core (PC) including PAS, GAF and PHY domains and an effector domain, usually a histidine kinase. The molecular mechanism that drives Pr/Pfr photoconversion is not well understood. Up to date, structures of intact Bphs remain unknown and only one Bph structure of complete PC has been solved. We recently obtained crystals of the unique Bph from Rhodopseudomonas palustris, RpBphP3 that diffracted to the maximum resolution of 2.8Å. P3 is naturally fluorescent and photoconverts between spectrally distinct red (Pr) and near-red (Pnr) states. The protein composed of PC only, crystallized as a dimer, bound to BV in the Pr state. The initial structure has been solved using Molecular Replacement with published P3 x-ray crystallographic structure composed of the PAS-GAF domains as the search model. Through site-directed mutagenesis we introduced single amino acid substitutions in the BV-binding pocket (P213G and I217T) and at the dimer interface (E315R, W323R and T142R) in order to optimize the fluorescence and photoactivity of the protein. Interestingly, mutants at the dimer interface showed more photoactivity compared to the wild type P3. Structural models addressing our results will be discussed.