Ultrasensitive probes of photoactive membrane proteins
Blue proteorhodopsin (BPR) and Sensory Rhodopsin II (SRII) 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 (pcFRET). We observed the photocycle in samples as small three molecules.
pcFRET is particularly suited to macromolecules which contain an endogenous chromophore that undergoes a chromatic shift. By labeling the macromolecule with a suitably chosen fluorophore, one can convert changes in the absorption of the chromophore to changes in the brightness of the fluorophore, thereby achieving a huge enhancement in detectability.
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Schematic of the mechanism of pcFRET. The fluorescent dye is quenched by the endogenous chromophore only when the absorption of the chromophore overlaps with the emission of the dye. |
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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 show the chromophore. |
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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