Biophysics and Physicobiology最新文献

TAT rhodopsin extracted from the marine bacterium SAR11 HIMB114 has a characteristic Thr-Ala-Thr motif and contains both protonated and deprotonated states of Schiff base at physiological pH conditions due to the low pK_a. Here, using solid-state NMR spectroscopy, we investigated the ¹³C and ¹⁵N NMR signals of retinal in only the protonated state of TAT in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (1'-rac-glycerol) (POPE/POPG) membrane at weakly acidic conditions. In the ¹³C NMR spectrum of ¹³C retinal-labeled TAT rhodopsin, the isolated 14-¹³C signals of 13-trans/15-anti and 13-cis/15-syn isomers were observed at a ratio of 7:3. ¹⁵N retinal protonated Schiff base (RPSB) had a significantly higher magnetic field resonance at 160 ppm. In ¹⁵N RPSB/λ_max analysis, the plot of TAT largely deviated from the trend based on the retinylidene-halide model compounds and microbial rhodopsins. Our findings indicate that the RPSB of TAT forms a very weak interaction with the counterion.

Direct observation of protein structural changes during ion transport in ion pumps provides valuable insights into the mechanism of ion transport. In this study, we examined structural changes in the light-driven sodium ion (Na⁺) pump rhodopsin KR2 on the sub-millisecond time scale, corresponding with the uptake and release of Na⁺. We compared the ion-pumping activities and transient absorption spectra of WT and the W215F mutant, in which the Trp215 residue located near the retinal chromophore on the cytoplasmic side was replaced with a Phe residue. Our findings indicated that atomic contacts between the bulky side chain of Trp215 and the C20 methyl group of the retinal chromophore promote relaxation of the retinal chromophore from the 13-cis to the all-trans form. Since Trp215 is conserved in other ion-pumping rhodopsins, the present results suggest that this residue commonly acts as a mechanical transducer. In addition, we measured time-resolved ultraviolet resonance Raman (UVRR) spectra to show that the environment around Trp215 becomes less hydrophobic at 1 ms after photoirradiation and recovers to the unphotolyzed state with a time constant of around 10 ms. These time scales correspond to Na⁺ uptake and release, suggesting evolution of a transient ion channel at the cytoplasmic side for Na⁺ uptake, consistent with the alternating-access model of ion pumps. The time-resolved UVRR technique has potential for application to other ion-pumping rhodopsins and could provide further insights into the mechanism of ion transport.

Since their discovery 21 years ago, channelrhodopsins have come of age and have become indispensable tools for optogenetic control of excitable cells such as neurons and myocytes. Potential therapeutic utility of channelrhodopsins has been proven by partial vision restoration in a human patient. Previously known channelrhodopsins are either proton channels, non-selective cation channels almost equally permeable to Na⁺ and K⁺ besides protons, or anion channels. Two years ago, we discovered a group of channelrhodopsins that exhibit over an order of magnitude higher selectivity for K⁺ than for Na⁺. These proteins, known as "kalium channelrhodopsins" or KCRs, lack the canonical tetrameric selectivity filter found in voltage- and ligand-gated K⁺ channels, and use a unique selectivity mechanism intrinsic to their individual protomers. Mutant analysis has revealed that the key residues responsible for K⁺ selectivity in KCRs are located at both ends of the putative cation conduction pathway, and their role has been confirmed by high-resolution KCR structures. Expression of KCRs in mouse neurons and human cardiomyocytes enabled optical inhibition of these cells' electrical activity. In this minireview we briefly discuss major results of KCR research obtained during the last two years and suggest some directions of future research.