Kwang-Hwan Jung

Salt bridge in the conserved His–Asp cluster in Gloeobacter rhodopsin contributes to trimer formation

Rhodopsin and Rhodopsin bind by molecular sieving (View interaction: 1, 2)

Spectroscopic and photochemical analysis of proteorhodopsin variants from the surface of the Arctic Ocean

Proteorhodopsin (PR), a retinal‐containing seven transmembrane helix protein, functions as a light‐driven proton pump. Using PCR, we isolated 18 PR variants originating from the surface of the Arctic Ocean. Their absorption maxima were between 517 and 546 nm at pH 7. One of the isolates turned out to be identical to GPR (green light‐absorbing proteorhodopsin) from Monterey Bay. Interestingly, 10 isolates had replaced a tyrosine in the retinal‐binding site (Tyr200 in GPR) with Asn. They showed a slower photocycle, more blue‐shifted absorption maxima at pH 10, and relatively larger ΔH and ΔS of activation of the transition between the O intermediate and the ground state compared to GPR.

Cyanobacterial phytochrome Cph2 is a negative regulator in phototaxis toward UV‐A

We investigated the wavelength dependence and photon‐fluence rate response relationship for phototaxis of wild‐type and a cyanobacterial phytochrome 2 (cph2) mutant in cyanobacterium Synechocystis sp. PCC 6803. Compared to wild‐type, the cph2 mutant exhibited maximal activity for positive phototaxis at the near‐UV spectral range. Two cysteine to serine substitutions in two chromophore‐binding domains showed a similar cph2 mutant phenotype under UV‐A. Epistasis of a pixJ mutation over a cph2 mutation implied that pixJ gene acts downstream of the cph2 gene with respect to UV‐A‐induced positive phototaxis. Therefore, we suggest that Cph2 is essential for the inhibition of positive phototaxis toward UV‐A.

Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum.

Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.