Confocal time resolved single-molecule spectroscopy using pulsed laser excitation and synchronized multi channel time correlated single photon counting (TCSPC) provides detailed information about the conformational changes of a biological motor in real time. We studied the formation of adenosine triphosphate, ATP, from ADP and phosphate by FoF1-ATP synthase. The reaction is performed by a stepwise internal rotation of subunits of the lipid membrane-embedded enzyme. Using fluorescence resonance energy transfer, FRET, we detected rotation of this biological motor by sequential changes of intramolecular distances within a single FoF1-ATP synthase. Prolonged observation times of single enzymes were achieved by functional immobilization to the glass surface. The stepwise rotary subunit movements were identified by Hidden Markov Models (HMM) which were trained with single-molecule FRET trajectories. To improve the accuracy of the HMM analysis we included the single-molecule fluorescence lifetime of the FRET donor and used alternating laser excitation to co-localize the FRET acceptor independently within a photon burst. The HMM analysis yielded the orientations and dwell times of rotary subunits during stepwise rotation. In addition, the action mode of bactericidal drugs, i.e. inhibitors of FoF1-ATP synthase like aurovertin, could be investigated by the time resolved single-molecule FRET approach.
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