Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Amati, Andrea Marco; Moning, Stefan Urs; Javor, Sacha; Schär, Sandra; Deutschmann, Sabina; Reymond, Jean-Louis; von Ballmoos, Christoph (2024). Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production. ACS synthetic biology, 13(4), pp. 1355-1364. American Chemical Society 10.1021/acssynbio.4c00058

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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.

Item Type:

Journal Article (Original Article)


08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP)

UniBE Contributor:

Amati, Andrea Marco, Moning, Stefan Urs, Javor, Sacha, Schär, Sandra, Deutschmann, Sabina Barbara, Reymond, Jean-Louis, von Ballmoos, Christoph


500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry
000 Computer science, knowledge & systems
600 Technology > 610 Medicine & health




American Chemical Society




Pubmed Import

Date Deposited:

04 Apr 2024 09:27

Last Modified:

20 Apr 2024 00:16

Publisher DOI:


PubMed ID:


Uncontrolled Keywords:

ATP synthesis energy conversion light-driven proton pumping liposomes membrane protein orientation synthetic biology





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