Many biological cofactors, such as light-absorbing chromophores in photoreceptors, contain a π-electron system and are planar molecules. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortions have been shown to be functionally important. Because the nonplanar structure makes the molecule chiral, Raman optical activity (ROA) provides a wealth of stereochemical information about the structural and conformational details of cofactors. The present study applied a near-infrared excited ROA to photoactive yellow protein, a blue light receptor. We successfully obtained the ROA spectra of the 4-hydroxycinnamyl chromophore embedded in a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

Many biological cofactors, such as light-absorbing chromophores in photoreceptors, are intrinsically planar molecules. A protein environment, however, causes structural distortions of the cofactor, and such structural changes can lead to a modulation of chemical properties of the cofactor to maximize its biological activity. Here, we investigate the active site structure of photoactive yellow protein (PYP), a blue light photoreceptor that contains a p-coumaric acid (pCA) chromophore, by a near-infrared excited Raman optical activity (ROA). Specifically, we measured the ROA spectra of PYP, whose chromophore is replaced with a locked pCA analogue. Furthermore, we show that a spectral analysis based on quantum mechanical/molecular mechanical (QM/MM) calculations of the whole protein molecule is useful to obtain structural information from the observed ROA spectra. The use of the near-infrared ROA combined with QM/MM calculations is a novel and generally applicable spectroscopic tool to study the chromophore distortions within a protein environment. SECTION: Biophysical Chemistry and Biomolecules

The flavin-binding BLUF domains are a class of blue-light receptors, and AppA is a representative of this family. Although the crystal and solution structures of several BLUF domains have already been obtained, there is a key uncertainty regarding the position of a functionally important tryptophan (Trp104 in AppA). In the first crystal structure of an N-terminally truncated BLUF domain of AppA133 (residues 17-133), Trp104 was found in close proximity to flavin (Trp(in)), whereas in a subsequent structure with an intact N-terminus AppA126 (residues 1-126), Trp104 was exposed to the solvent (Trp(out)). A recent study compared spectroscopic properties of AppA126 and AppA133 and claimed that the Trp(in) conformation is an artifact of N-terminal truncation in AppA133. In this study, we compared the flavin vibrational spectra of AppA126 and AppA133 by using near-infrared excited Raman spectroscopy. In addition, the conformations as well as the environments of Trp104 were directly monitored by ultraviolet resonance Raman spectroscopy. These studies demonstrate that the N-terminal truncation does not induce the conformational switch between Trp(in) and Trp(out).

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.

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