Actinorhodopsin: an efficient and robust light-driven proton pump for bionanotechnological applications.

Actinorhodopsins are encoded by a distinct group of microbial rhodopsin (MR) genes predominant in non-marine actinobacteria. Despite their role in the global energy cycle and potential for bionanotechnological applications, our understanding of actinorhodopsin proteins is limited. Here, we characterized the actinorhodopsin RlActR from the freshwater actinobacterium Rhodoluna lacicola, which conserves amino acid residues critical for light-driven proton pumping found in MRs. RlActR was efficiently overexpressed in Escherichia coli in milligram amounts and isolated with high purity and homogeneity. The purified RlActR absorbed green light and its primary proton acceptor exhibited a mildly acidic apparent pKa. Size-exclusion chromatography of RlActR purified in the relatively mild and harsh detergents 5-cyclohexyl-1-pentyl-β-D-maltoside and n-octyl-β-D-glucopyranoside revealed highly homogeneous oligomers and no disruption into monomers, indicating significant robustness of the RlActR oligomer. Cryo-electron microscopy and 2D classification of protein particles provided a projection structure identifying the oligomeric state of RlActR as a pentamer. Efficient establishment of a proton gradient across lipid membranes upon light illumination was demonstrated using RlActR-overexpressing E. coli cells and reconstituted RlActR proteoliposomes. In summary, these features make RlActR an attractive energizing building block for the bottom-up assembly of molecular systems for bionanotechnological applications.