Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.     

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.     

Induction of DNA damage, including abasic sites, in plasmid DNA by carbon ion and X-ray irradiation

DNA from plasmid pUC18 was irradiated with low-LET (13 keV/μm) or high-LET (60 keV/μm) carbon ions or X-rays (4 keV/μm) in solutions containing several concentrations of Tris (0.66–200 mM) to determine the yield of abasic (AP) sites and the effect of scavenging capacity. The yield of AP sites, detected as single-strand breaks (SSB) after digestion with E. coli endonuclease IV (Nfo), was compared with that of SSB and base lesions. At higher concentrations of Tris, the yields of single or clustered AP sites were significantly lower than those of single or clustered base lesions. The relative yields of single AP sites and AP clusters were less than 10 and 7 %, respectively, of the total damage produced at a scavenger capacity mimicking that in cells. The dependence of the yield of AP sites on scavenging capacity was similar to that of prompt strand breaks. The ratios of the yield of isolated AP sites to that of SSB induced by carbon ion or X-ray irradiation were relatively constant at 0.45 ± 0.15 over the tested range of scavenger capacity, although the ratio of SSB to double-strand breaks (DSB) showed the characteristic dependence on both scavenging capacity and radiation quality. These results indicate that the reaction of water radiolysis products, presumably OH radicals, with the sugar-phosphate moieties in the DNA backbone induces both AP sites and SSB with similar efficiency. Direct ionization of DNA is notably more involved in the production of DSB and base lesion clusters than in the production of AP site clusters.     

Genetic and family structure in a group of 165 common bottlenose dolphins caught off the Japanese coast

The biological and genetic structure of common bottlenose dolphins (Tursiops truncatus) that migrate seasonally near Japan remains largely unknown. We investigated the genetic and family structure in a group of 165 common bottlenose dolphins caught off the coast of Japan using mitochondrial DNA (mtDNA) and 20 microsatellite DNA markers. Phylogenetic analysis of the mtDNA control region sequences suggested that the dolphins were related more closely to oceanic types from Chinese waters than other geographic regions. The information on sex, sexual maturation and age together with the genetic markers revealed a strong likelihood for 37 familial relationships related mostly to maternity and an under‐representation of juvenile female offspring. The maternal dolphins had a similar offspring‐birth interval as the coastal types from North Atlantic Ocean, but a slightly younger first‐progeny age. The sex bias in the captured group was particularly marked towards an over‐representation of males among the young and immature dolphins, whereas the mature adults had an equal number of males and females. These results should be useful for future comparative biological, genetic and evolutionary investigations of bottlenose dolphins from the North Pacific Ocean with those from other regions.     

Crystal structure of deltarhodopsin‐3 from Haloterrigena thermotolerans

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.