title: Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.
abstract: The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H2MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H2MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H2MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼104s-1. The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science , Rehovot 76100, Israel.
authors.forename: Menahem
authors.initials: M
authors.lastname: Pirchi
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Roman
authors.initials: R
authors.lastname: Tsukanov
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science , Rehovot 76100, Israel.
authors.forename: Rashid
authors.initials: R
authors.lastname: Khamis
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Toma E
authors.initials: TE
authors.lastname: Tomov
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Yaron
authors.initials: Y
authors.lastname: Berger
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Dinesh C
authors.initials: DC
authors.lastname: Khara
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Hadas
authors.initials: H
authors.lastname: Volkov
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science , Rehovot 76100, Israel.
authors.forename: Gilad
authors.identifier: 0000-0003-1837-9779
authors.initials: G
authors.lastname: Haran
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Eyal
authors.initials: E
authors.lastname: Nir