Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET

N. Zarrabi; T. Heitkamp; J.-C. Greie; M. Börsch

doi:10.1117/12.768262

21 February 2008 Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET

N. Zarrabi, T. Heitkamp, J.-C. Greie, M. Börsch

Proceedings Volume 6862, Single Molecule Spectroscopy and Imaging; 68620M (2008) https://doi.org/10.1117/12.768262
Event: SPIE BiOS, 2008, San Jose, California, United States

Abstract

Conformational changes of single proteins are monitored in real time by Förster-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.

Citation Download Citation

N. Zarrabi, T. Heitkamp, J.-C. Greie, and M. Börsch "Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET", Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 68620M (21 February 2008); https://doi.org/10.1117/12.768262

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