Retinal-based Proton Pumping in the Near Infrared

Proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) are retinal-based light-driven proton pumps which absorb visible light (maxima 520-540 nm). Shifting the action spectra of these proton pumps beyond 700 nm would generate new prospects in optogenetics, membrane sensor technology, and complementation of oxygenic phototrophy. We thereby investigated the effect of red-shifting analogs of retinal, combined with red-shifting mutations, on the spectral properties and pump activity of the resulting pigments. We investigated a variety of analogs, including many novel ones. One of the novel analogs we tested, 3-methylamino-16-nor-1,2,3,4-didehydroretinal (MMAR), produced exciting results. This analog red-shifted all rhodopsin variants tested, accompanied by a strong broadening of the absorbance band, tailing out to 850-950 nm. In particular, MMAR showed a strong synergistic effect with the PR-D212N,F234S double mutant, inducing an astonishing 200 nm red-shift in the absorbance maximum. To our knowledge, this is by far the largest red-shift reported for any retinal protein. Very importantly, all MMAR containing holoproteins are the first rhodopsins retaining significant pump activity under near infra-red illumination (730 nm LED). Such MMAR-based rhodopsin variants present very promising opportunities for further synthetic biology modification and for a variety of biotechnological and biophysical applications.