Engineering a Chemical Switch into the Light-driven Proton Pump Proteorhodopsin by Cysteine Mutagenesis and Thiol Modification.
For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system.
Institute of Biochemistry and Molecular Medicine, University of Bern, 3012, Bern, Switzerland.
Daniel
D
Harder
Institute of Biochemistry and Molecular Medicine, University of Bern, 3012, Bern, Switzerland.
Stephan
S
Hirschi
Institute of Biochemistry and Molecular Medicine, University of Bern, 3012, Bern, Switzerland.
Zöhre
Z
Ucurum
Department of Chemistry, University of Basel, 4056, Basel, Switzerland.|Department of Biosystems Science and Engineering, ETH Zürich, 4058, Basel, Switzerland.
Roland
R
Goers
Department of Chemistry, University of Basel, 4056, Basel, Switzerland.
Wolfgang
W
Meier
Department of Biosystems Science and Engineering, ETH Zürich, 4058, Basel, Switzerland.
Daniel J
DJ
Müller
Institute of Biochemistry and Molecular Medicine, University of Bern, 3012, Bern, Switzerland. dimitrios.fotiadis@ibmm.unibe.ch.
Dimitrios
D
Fotiadis
light-driven proton pumps; membrane proteins; protein engineering; proteorhodopsin; synthetic biology