Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium

Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli c ells e xpressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+- pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the alltrans retinal conformer. (ii) Time- dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+- pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.

Automated summary

In this study, a newly identified rhodopsin called Bellilinea Na+-pumping rhodopsin (BeNaR) was characterized. BeNaR has a significantly higher Na+-pumping activity compared to known Na+-pumping rhodopsin KR2. The findings provide a useful model for studying ion transporters and optogenetic tools.