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The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly 10-fold increase in red-light emission, the second is not emissive. Thus, the W76S/Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multiconfigurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming