title: Inter-dye distance distributions studied by a combination of single-molecule FRET-filtered lifetime measurements and a weighted accessible volume (wAV) algorithm.
abstract: Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers.
authors.affiliation: Physikalisches Institut (IA), RWTH Aachen University, Otto-Blumenthal-Straße, 52074 Aachen, Germany. hoefig@physik.rwth-aachen.de.
authors.forename: Henning
authors.initials: H
authors.lastname: Höfig
authors.affiliation: Institute of Complex Systems (ICS-5), Molecular Biophysics, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany. m.gabba@fz-juelich.de.
authors.forename: Matteo
authors.initials: M
authors.lastname: Gabba
authors.affiliation: Institute of Complex Systems (ICS-2), Theoretical Soft Matter and Biophysics, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany. s.poblete@fz-juelich.de.
authors.forename: Simón
authors.initials: S
authors.lastname: Poblete
authors.affiliation: Physikalisches Institut (IA), RWTH Aachen University, Otto-Blumenthal-Straße, 52074 Aachen, Germany. kempe@physik.rwth-aachen.de.
authors.forename: Daryan
authors.initials: D
authors.lastname: Kempe
authors.affiliation: Physikalisches Institut (IA), RWTH Aachen University, Otto-Blumenthal-Straße, 52074 Aachen, Germany. fitter@physik.rwth-aachen.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fitter
mesh_terms: D000465:Algorithms; D020029:Base Pairing; D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008956:Models, Chemical
chemical_list: D004396:Coloring Agents; D005456:Fluorescent Dyes; D004247:DNA