Fluorescence resonance energy transfer in single enzyme molecules with a quantum dot as donor

H + -ATPsynthases couple a transmembrane proton transport with ATP synthesis and ATP hydrolysis. Previously, the relative subunit movement during this process has been measured by fluorescence resonance energy transfer (FRET) between two organic fluorophores covalently bound to different subunits. To improve the photophysical stability, a luminescent CdSe/ZnS nanocrystal (quantum dot) was bound to the enzyme and an organic fluorophore, Alexa568, was used as fluorescence acceptor. Single-molecule spectroscopy with the membrane integrated labeled H + -ATPsynthase was carried out. Single-pair FRET indicates three different conformations of the enzyme. During ATP hydrolysis relative intramolecular subunit movements are observed in real time.