1P239 Active Site Structure of Photoactive Yellow Protein with a Locked Chromophore Analog Revealed by Near Infrared Raman Optical Activity(18A. Photobiology: Vision & Photoreception,Poster)

Abstract

Microbial rhodopsins are photoactive proteins that use retinal molecule as the photoactive center and have been studied intensively as an excellent model system for structural biology. We have identified the new microbial rhodopsin genes from halorubrum species isolated from Ejinoor salt lake in Inner Mongolia. Pairs of PCR primers targeted bacteriorhodopsin (bR) gene or phoborhodopsin (pR) gene were made. We got the full-length sequence of bR-like gene by genome walking. The deduced amino acid sequence has 87% of identity with that of H.s. All the amino acids important for the proton pumping are conserved. The cloned pR homolog showed 60% of identity with that of H.s. in deduced amino acids sequence. 1P237ニワトリクリプトクロム 4 の光反応特性の解析 Cryptochromes (CRYs) function as blue light photoreceptors in bacteria, plants, invertebrates, and non-mammalian vertebrates. In order to understand molecular properties of CRYs in lower vertebrates, we focused on zebrafish cryptochromes, the physiological function of which remains elusive. We expressed a HAT-tagged zebrafish CRY1a (zCRY1a) in Saccharomyces cerevisiae exogeneously and zCRY1a was purified by using immobilized metal ion affinity chromatography. The purified zCRY1a protein did not show characteristic spectral feature indicating flavin binding, but it bound with flavin adenine dinucleotide (FAD) when FAD was added. This novel system will enable us to investigate photic reaction of zCRY1a by UV-visible spectroscopy. Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors and contain a linear tetrapyrrole (bilin) chromophore. RcaE from Fremyella diplosiphon has been shown to undergo a reversible photoconversion between a green-absorbing Pg state and a red-absorbing Pr state. A previous pH titration experiment suggested that the bilin chromophore is deprotonated in Pg but protonated in Pr (1).Here we have measured the resonance Raman spectra of RcaE in Pg and Pr states and provide spectroscopic evidence that the Pg/Pr photoconversion is associated with a protonation/deprotonation of the bilin chromophore.