posted on 2024-04-03, 19:35authored byAndrea
Marco Amati, Stefan Urs Moning, Sacha Javor, Sandra Schär, Sabina Deutschmann, Jean-Louis Reymond, Christoph von Ballmoos
Adenosine triphosphate (ATP)-producing modules energized
by light-driven
proton pumps are powerful tools for the bottom-up assembly of artificial
cell-like systems. However, the maximum efficiency of such modules
is prohibited by the random orientation of the proton pumps during
the reconstitution process into lipid-surrounded nanocontainers. Here,
we overcome 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, we 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
are verified by monitoring vectorial proton pumping and membrane potential
generation. In conjunction with ATP synthase, highly efficient ATP
production is energized by the inwardly pumping population. In comparison
to other light-driven ATP-producing modules, the uniform orientation
allows for maximal rates at economical protein concentrations. The
presented technology is highly customizable and not limited to light-driven
proton pumps but 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.