Spectral Shift FRET Assay and its Applications for Studying the Dynamics of Proteorhodopsin

Abstract

We present a highly sensitive method of studying the dynamics of photosensitive membrane protein proteorhodopsin (PR), by using spectral shift FRET. In normal FRET, the rate of energy transfer depends on the spatial separation of donor and acceptor. In spectral shift FRET, the rate of energy transfer depends on the spectral separation, which varies in response to changes in the chemical environment of one of the chromophores. Our method is particularly suited to macromolecules that contain an endogenous chromophore that undergoes chromatic shifts, in which case only a single fluorescent label is required. The label serves as a fluorescence donor, and the endogenous chromophore serves as an environmentally sensitive quencher.Proteorhodopsin found in marine bacterioplankton is a membrane protein that functions as a light-driven proton pump, converting light energy into chemical energy by creating a proton motive force across the bacterial membrane. The retinal chromophore undergoes dramatic spectral shifts during the photocycle. Bodipy-Texas Red (TR) was incorporated into a blue-absorbing variant of PR on the cytoplasmic side through a single endogenous cysteine (CYS116). The photocycle was initiated by a 50 ms pulse at 490 nm, and the ensuing dynamics were probed by measuring the fluorescence quantum yield of Bodipy-TR (excitation at 600 nm, detection at 650-700 nm). The signal from a single PR-containing 340 nm lipid vesicle was sufficient to monitor the dynamics of the photocycle, thereby providing a highly sensitive method to monitor microbial rhodopsins. In future studies, a vesicle containing a single PR molecule will be trapped using an Anti-Brownian Electrokinetic trap, and fluctuations in the dynamics of PR will be observed using spectral shift FRET. The technique of spectral shift FRET provides an important new tool for studies of photosensitive proteins.