Engineering a Chemical Switch into the Light‐driven Proton Pump Proteorhodopsin by Cysteine Mutagenesis and Thiol Modification

Abstract

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.

A Light‐Responsive Metal–Organic Framework Hybrid Membrane with High On/Off Photoswitchable Proton Conductivity

“…This approach might provide a platform for creating emerging smart protonic solids with potential applications in the remote-controllable chemical sensors or protonconducting field-effect transistors.Many biological structures in nature can manipulate the substance transportation and signal transduction with the unique responsiveness to external stimuli, especially light. [1] An intriguing example is provided by bacteriorhodopsin (bR), a membrane protein known as light-activated proton pumps, which harvests energy from the light and generates proton gradients across the membrane to drive ATP production. [2] In contrast to biological materials, the artificial ones with better stability and modularity can be applied as biomimetics of light-driven proton pump and work under tough conditions.…”

Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics

“…Time-resolved advanced spectroscopy and X-ray crystallography, site-directed alteration ( i.e . substitution of a natural or unnatural amino acid) or the novel chemical mutagenesis approaches [104–106], and synthetic techniques for mechanism-based probes are expected to yield fundamental structural and chemical insights into O 2 activation and insertion for mechanistic enzymology of the non-heme Fe dioxygenases.…”

Light-Driven Biocatalysis in Liposomes and Polymersomes: Where Are We Now?

“…A sophisticated method to avoid the undesired proton pumping activity of wrongly oriented proton pumps is the selective deactivation of proteins that were integrated in non-physiological orientation. D. Fotiadis and coworkers developed a rationally designed PR mutant with an engineered on/off switch based on the reversible chemical modification of an introduced cysteine residue that allows for the selective deactivation of wrongly oriented PR molecules [75]. A clear disadvantage of this method is the net-loss of proton pumping activity which is undesirable in view of the laborious isolation and reconstitution of proton pumps.…”