Figure 1.
Pylogenetic tree and alignment.
(a) Phylogenetic tree of 22 microbial opsin sequences, representing phylogenetic relationship between Gloeobacter rhodopsin and related proteins from archaea to algae. Analysis was conducted by ClustalW. A dotted line on a phenogram indicates a negative branch length, a common result of averaging by the DNA STAR software. GR: Gloeobacter rhodopsin (accession number; NP_923144). Red23 and REDr6a5a2 were collected from Red Sea. GRP/BPR: green/blue-absorbing PRs. PalE6 was collected from Antarctic ocean. RhodospR: rhodopsin from Rhodospirillales sp. XR: Xanthorhodopsin from Salinibacter ruber. FulviR: rhodopsin from Fulvimarina. ArchaerR1: Archaerhodopsin 1. PYR: rhodopsin from Pyrocystis lunula. BR: Halobacterium salinarum bacteriorhodopsin. RoseiR: rhodopsin from Roseiflexus sp.MarinoR: rhodopsin from Marinobacter. MethyloR: rhodopsin from Methylophilales.LR: rhodopsin from Leptosphaeria maculans. NR: rhodopsin from Neurospora crassa. AR: rhodopsin from Acetabularia acetabulum. SRI: H. salinarum sensory rhodopsin I. NpSRII: Natronomonas pharaonis sensory rhodopsin II. ASR: sensory rhodopsin from Anabaena (Nostoc) sp.CSOA: Chlamydomonas reinhardtii sensory rhodopsin A. (b) Alignment of the sequences of GR, XR, PR, RoseiR, MarinoR, MethyloR and BR. GR contains the functionally important residues for proton transport, including homologues of Arg82, Asp85, Trp86, Asp96, Trp182, Tyr185, Asp212, and Lys216, with some of the numbering and helical segments (marked by arrows) for BR. There are 22 residues (marked with black boxes) common to all seven proteins (in one case, Asp/Glu substitutions). The Asp121 (Asp85 in BR) should be the proton acceptor of the retinal Schiff base. The Glu132 (Asp96 in BR) may be the proton donor.
Figure 2.
Absorption spectra and titration curves of GR and its mutants.
Insets show photos of purified WT and mutant GR in 50 mM Tris (pH 7), 150 mM NaCl, and 0.02% DDM. The pH titration curves indicate the pH dependence of the absorption maxima of broad spectral range. The pKa values are shown in the table 1.
Table 1.
Absorption maximum and pKa value of wild-type and various GR mutants.
Figure 3.
Proton pumping activity and photocycle kinetics.
(a) Light induced proton fluxes in E. coli vesicles containing wild type and mutant GR. Initial pH values were adjusted to7. The buffering capacity was calibrated and the pH changes were recalculated into proton concentrations. (b) Photocycle kinetics of the wild-type GR measured in polyacrylamide gel-encased, DM-treated membranes at 22°C, in 100 mM NaCl, 50 mM KH2PO4, pH 8. This figure gives overallcharacteristics of the photocycle with the differential absorption measured at 620 nm for theK and O intermediates, 410 nm for the M intermediate, 460 nm for the L intermediate, and 560 nm for the parent state and the N intermediate. See the full photocycle description in [20].
Figure 4.
Immunoblot and spectroscopy analysis.
(a) Immunoblot analysis of the Gloeobacter cells and the His-tagged GR protein. The immunoblot used anti-GR antibody for the membrane protein fraction of Gloeobacter cells and anti-His-tag antibody for the protein expressed in E. coli. The whole cells (C) were sonicated and lysates were ultracentrifuged. The lysates were separated into supernatant (A: soluble protein) and pellet (B: membrane protein). D: purified GR from E. coli. (b) Absorption spectra of intact cells and membranes of G. violaceus. The spectrum of G. violaceus is shown with solid line. Chl a (chlorophyll a), Car (carotenoid), PE (phycoerythrin), GR, and PC (phycocyanin) are marked by the arrows. The spectrum of G. violaceus membrane is shown by the dashed line. The spectrum of purified GR in 0.02% DDM, isolated from E. coli is shown by dotted line.
Figure 5.
Action spectra for light-driven proton translocation in Gloeobacter cells (solid line).
Sphaeroplasts of Gloeobacter cells were illuminated through the filters transmitting at 700, 650, 600, 550, 500, 450, and 400 nm. The efficiency of the sphaeroplasts (after the photosynthesis inhibition by DCMU treatment (5×10−5mol l−1)) was most efficient at 550 nm (dashed line). Integrated H+ pumping amount is presented by blank column. Concentration of synthesized ATP is presented by slashed column.
Figure 6.
Schematic model of energy production in Gloeobacter.
The photosynthetic apparatus of Gloeobacter is reproduced from [51].