title: Temperature-cycle single-molecule FRET microscopy on polyprolines.
abstract: Accessing the microsecond dynamics of a single fluorescent molecule in real time is difficult because molecular fluorescence rates usually limit the time resolution to milliseconds. We propose to apply single-molecule temperature-cycle microscopy to probe molecular dynamics at microsecond timescales. Here, we follow donor and acceptor signals of single FRET-labeled polyprolines in glycerol to investigate their conformational dynamics. We observe a steady-state FRET efficiency distribution which differs from theoretical distributions for isotropically orientated fluorescent labels. This may indicate that the orientation of fluorescent labels in glycerol is not isotropic and may reflect the influence of the dye linkers. With proper temperature-cycle parameters, we observed large FRET changes in long series of cycles of the same molecule. We attribute the main conformational changes to reorientations of the fluorescent labels with respect to the oligopeptide chain, which take place in less than a few microseconds at the highest temperature of the cycle (250 K). We were able to follow the FRET efficiency of a particular construct for more than 2000 cycles. This trajectory displays switching between two conformations, which give rise to maxima in the FRET efficiency histogram. Our experiments open the possibility to study biomolecular dynamics at a time scale of a few microseconds at the single-molecule level.
authors.affiliation: Molecular Nano-Optics and Spins, Leiden Institute of Physics, Leiden University, Leiden, The Netherlands.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Yuan
authors.forename: Ted
authors.initials: T
authors.lastname: Xia
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
authors.forename: Michel
authors.initials: M
authors.lastname: Orrit
mesh_terms: D003080:Cold Temperature; D031541:Fluorescence Resonance Energy Transfer; D006358:Hot Temperature; D018613:Microscopy, Confocal / Q000379:methods*; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry*; D013696:Temperature
chemical_list: D010455:Peptides; C011083:polyproline