title: Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models.
abstract: Single-molecule Förster resonance energy (smFRET) transfer has become a powerful tool for observing conformational dynamics of biological macromolecules. Analyzing smFRET time trajectories allows to identify the state transitions occuring on reaction pathways of molecular machines. Previously, we have developed a smFRET approach to monitor movements of the two nucleotide binding domains (NBDs) of P-glycoprotein (Pgp) during ATP hydrolysis driven drug transport in solution. One limitation of this initial work was that single-molecule photon bursts were analyzed by visual inspection with manual assignment of individual FRET levels. Here a fully automated analysis of Pgp smFRET data using hidden Markov models (HMM) for transitions up to 9 conformational states is applied. We propose new estimators for HMMs to integrate the information of fluctuating intensities in confocal smFRET measurements of freely diffusing lipid bilayer bound membrane proteins in solution. HMM analysis strongly supports that under conditions of steady state turnover, conformational states with short NBD distances and short dwell times are more populated compared to conditions without nucleotide or transport substrate present.
authors.affiliation: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University Jena, 07743 Jena, Germany; 3rd Institute of Physics, University of Stuttgart, 70550 Stuttgart, Germany.
authors.forename: Nawid
authors.initials: N
authors.lastname: Zarrabi
authors.affiliation: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University Jena, 07743 Jena, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ernst
authors.affiliation: Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
authors.forename: Brandy
authors.initials: B
authors.lastname: Verhalen
authors.affiliation: Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilkens
authors.affiliation: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University Jena, 07743 Jena, Germany; 3rd Institute of Physics, University of Stuttgart, 70550 Stuttgart, Germany. Electronic address: michael.boersch@med.uni-jena.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Börsch
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry*; D000465:Algorithms; D000818:Animals; D001692:Biological Transport; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D008390:Markov Chains; D018613:Microscopy, Confocal; D008958:Models, Molecular; D011487:Protein Conformation; D012996:Solutions
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D008081:Liposomes; D012996:Solutions
keywords: Hidden Markov model; P-glycoprotein; Protein dynamics; Single-molecule FRET
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