Ultrasensitive Measurements of Microbial Rhodopsin Photocycles Using Photochromic FRET

Microbial rhodopsins are an important class of light‐activated transmembrane proteins whose function is typically studied on bulk samples. Herein, we apply photochromic fluorescence resonance energy transfer to investigate the dynamics of these proteins with sensitivity approaching the single‐molecule limit. The brightness of a covalently linked organic fluorophore is modulated by changes in the absorption spectrum of the endogenous retinal chromophore that occur as the molecule undergoes a light‐activated photocycle. We studied the photocycles of blue‐absorbing proteorhodopsin and sensory rhodopsin II (SRII). Clusters of 2–3 molecules of SRII clearly showed a light‐induced photocycle. Single molecules of SRII showed a photocycle upon signal averaging over several illumination cycles. Blue proteorhodopsin and Sensory Rhodopsin II are transmembrane proteins with a light‐induced photocycle. The retinal chromophore undergoes dramatic shifts in absorption spectrum during the photocycle, but absorption measurements lack sensitivity to detect these shifts in microscopic samples. We generated a fluorescence‐based readout of the photocycle by appending a small organic fluorophore to the protein. The fluorescence was quenched during phases of the photocycle in which the absorption spectrum of the retinal overlapped with the emission spectrum of the dye, an effect called photochromic fluorescence resonance energy transfer. We observed the photocycle in samples as small three molecules.