Microbial rhodopsins are photoactive proteins that use a retinal molecule as the photoactive center. Because of structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In this study, a halophilic archaea that has three microbial rhodopsin-type genes in its genome was isolated from Ejinoor salt lake in Inner Mongolia of China. A sequence of 16S rRNA showed that the strain belongs to Halorubrum genus and named Halorubrum sp. ejinoor (He). The translated amino acid sequences of its microbial rhodopsin-type genes suggest that they are homologs of archaerhodopsin (HeAR), halorhodopsin (HeHR) and sensory rhodopsin II (HeSRII). The mRNAs of three types of genes were detected by RT-PCR and their amounts were investigated by Real-Time RT-PCR. The amount of mRNA of HeSRII was the smallest and the amounts of of HeAR and HeHR were 30 times and 10 times greater than that of HeSRII. The results of light-induced pH changes suggested the presence of a light-driven proton pump and a light-driven chloride ion pump in the membrane vesicles of He. Flash induced absorbance changes of the He membrane fraction indicated that HeAR and HeHR are photoactive and undergo their own photocycles. This study revealed that three microbial rhodopsin-type genes are all expressed in the strain and at least two of them, HeAR and HeHR, are photochemically and physiologically active like BR and HR of Halobacterium salinarum, respectively. To our knowledge, this is the first report of physiological activity of HR-homolog of Halorubrum species.
Aspartic acid 103 (D103) of sensory rhodopsin II from Halobacterium salinarum (HsSRII, or also called phoborhodopsin) corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue is functionally important in BR. This work reveals that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can cause large changes in HsSRII photocycle. These changes include (1) shortened lifetime of the M intermediate in the following order: the wild-type > D103N > D103E; (2) altered decay pathway of a 13-cis O-like species. The 13-cis O-like species, tentatively named Px, was detected in HsSRII photocycle. Px appeared to undergo branched reactions at 0°C, leading to a recovery of the unphotolyzed state and formation of a metastable intermediate, named P370, that slowly decayed to the unphotolyzed state at room temperature. In wild-type HsSRII at 0°C, Px mainly decayed to the unphotolyzed state, and the decay reaction toward P370 was negligible. In mutant D103E at 0°C, Px decayed to P370, while the recovery of the unphotolyzed state became unobservable. In mutant D103N, the two reactions proceeded at comparable rates. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII.
We describe the measurement of surface acoustic wave (SAW) velocities to identify five odorant molecules by using a SAW device system. We derive a new frequency equation for SAWs propagating in the SAW device system with Cynops pyrrhogaster lipocalin (Cp-Lip1) protein, in order to identify five odorant molecules, R-limonene (R-Lim), ethyl butyrate (Eth), 2-isobutylthiazole (Iso), benzophenone (Ben), and 2-acetylthiazole (Ace). We developed a method to identify these odorant molecules combined with the Cp-Lip1 odorant-binding protein. Our frequency equation can satisfactorily predict different odorant molecules in the Cp-Lip1 SAW device. Moreover, our data suggest that the propagation velocity of the SAWs mostly relate to the density and concentration of the Cp-Lip1 odorant molecule mixtures. At the same sample concentration, the propagation velocity depends on the density. For the same odorant molecule, the propagation velocity decreases with increasing concentration.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.