A blue-shifted light-driven proton pump for neural silencing.

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λmax = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.