3.F.4.  The Nanoscale Light-driven ATP Synthesis (NLAS) Family 

Overcoming protein orientation mismatch enables efficient nanoscale light-driven ATP synthesis (Amati et al. 2024).  The maximal efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Amati et al. 2024 overcame this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain, guiding orientation during insertion into preformed liposomes. In the second scenario, they developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations  were verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production was energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allowed for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but is applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest (Amati et al. 2024).