Quantum dots for single-pair fluorescence resonance energy transfer in membrane- integrated EFoF1.
ABSTRACT spFRET (single-pair fluorescence resonance energy transfer) with organic fluorophores has been used to demonstrate rotation of the subunits gamma and epsilon in membrane-integrated FoF1 during proton transport-coupled ATP synthesis. Owing to the high light intensities used in single-molecule spectroscopy, organic fluorophores show a high probability for photobleaching. Luminescent CdSe/ZnS nanocrystals with a hydrophilic shell have been covalently bound to FoF1 either to the stator subunit b or to the rotor subunit c. TIRFM (total internal reflection microscopy) shows that covalent binding of the QD (quantum dot) via cysteine to FoF1 leads to a significant decrease in the blinking probability in the microsecond-to-second time range. This effect allows the observation of subunit movements in an extended time range. If the QD is bound to the rotor subunit c, the fluorescence anisotropy shows fluctuations in the presence of ATP, in contrast with the constant anisotropy observed in the absence of ATP.