Conformational changes of single proteins are monitored in real time byForster-type resonance energy transfer, FRET. Two different fluorophores haveto be attached to those protein domains, which move during function. Thedistance between the fluorophores is measured by relative fluorescenceintensity changes of FRET donor and acceptor fluorophore, or by fluorescencelifetime changes of the FRET donor. The fluorescence spectrum of a single FRETdonor fluorophore is influenced by local protein environment dynamics causingapparent fluorescence intensity changes on the FRET donor and acceptor detectorchannels. To discriminate between those spectral fluctuations anddistance-dependent FRET, alternating pulsed excitation schemes (ALEX) haverecently been introduced which simultaneously probe the existence of a FRETacceptor fluorophore. Here we employ single-molecule FRET measurements to amembrane protein. The membrane-embedded KdpFABC complex transports potassiumions across a lipid bilayer using ATP hydrolysis. Our study aims at theobservation of conformational fluctuations within a single P-type ATPasefunctionally reconstituted into liposomes by single-molecule FRET and analysisby Hidden-Markov-Models.
展开▼
