Ultrasensitive probes of photoactive membrane proteins  

Halil Bayraktar, Alex Fields, Joel Kralj

Spectral Shift FRET (ssFRET) is a highly sensitive method to probe the dynamics of photosensitive membrane proteins. In normal FRET, the rate of energy transfer depends on the spatial separation of donor and acceptor. In ssFRET, the rate of energy transfer depends on the spectral separation of a donor and an environmentally sensitive acceptor. This method is particularly suited to macromolecules which contain an endogenous chromophore that undergoes a chromatic shift, in which case only a single fluorescent label is required. The label serves as a fluorescence donor, and the endogenous chromophore serves as the dynamic quencher in response to photon activation or environmental changes.

Proteorhodopsin (PR) and Sensory Rhodopsin II (SRII), found in marine plankton and haloarchea, respectively, are light sensitive membrane proteins that convert light into chemical energy by creating a proton-motive force across the membrane. The retinal chromophore undergoes dramatic spectral shifts during the photocycle. We attached Alexa dyes to single-cysteine mutants of PR and SRII. The photocycle was initiated by flashes of blue light, and the ensuing dynamics were probed by measuring the fluorescence quantum yield of the Alexa dyes. 100 nm size vesicle containing a few copies of membrane proteins were sufficient to monitor the dynamics of the photocycle, thereby providing a highly sensitive method to monitor microbial rhodopsins. The technique of Spectral Shift FRET provides an important new tool for studies of photosensitive proteins.

Schematic of the mechanism of ssFRET. The fluorescent dye is quenched by the endogenous chromophore only when the absorption of the chromophore overlaps with the emission of the dye.

Here is a cartoon showing a molecule of SRII in a lipid bilayer, with an attached fluorophore (red star), and the endogenous retinal molecule (green line in the middle of the molecule). Some of the helices have been cut away to allow visualization of the chromophore.

 

Typical data for SRII shows a decrease in the fluorescence after a flash of blue light, followed by a slower rise in the fluorescence. The decrease is due to a transition in the retinal to a state whose absorption overlaps with the emission of the fluorophore (Alexa 546); the recovery occurs when the retinal returns to its ground state, which does not have spectral overlap with the fluorophore.

 

Documentation:

• Halil Bayraktar poster presentation at 2009 Biophysical Society Meeting, March 1, 2009