  Conformational changes of single proteins are monitored in real time by
Forster-type resonance energy transfer, FRET. Two different fluorophores have
to be attached to those protein domains, which move during function. The
distance between the fluorophores is measured by relative fluorescence
intensity changes of FRET donor and acceptor fluorophore, or by fluorescence
lifetime changes of the FRET donor. The fluorescence spectrum of a single FRET
donor fluorophore is influenced by local protein environment dynamics causing
apparent fluorescence intensity changes on the FRET donor and acceptor detector
channels. To discriminate between those spectral fluctuations and
distance-dependent FRET, alternating pulsed excitation schemes (ALEX) have
recently been introduced which simultaneously probe the existence of a FRET
acceptor fluorophore. Here we employ single-molecule FRET measurements to a
membrane protein. The membrane-embedded KdpFABC complex transports potassium
ions across a lipid bilayer using ATP hydrolysis. Our study aims at the
observation of conformational fluctuations within a single P-type ATPase
functionally reconstituted into liposomes by single-molecule FRET and analysis
by Hidden-Markov-Models.
