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Molecules 2014, 19(12), 19269-19291; doi:10.3390/molecules191219269

Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV) Algorithm

1,2,†
,
2,†,* , 3,†
,
1
and
1,2,*
1
Physikalisches Institut (IA), RWTH Aachen University, Otto-Blumenthal-Straße, 52074 Aachen, Germany
2
Institute of Complex Systems (ICS-5), Molecular Biophysics, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany
3
Institute of Complex Systems (ICS-2), Theoretical Soft Matter and Biophysics, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 23 August 2014 / Revised: 3 November 2014 / Accepted: 6 November 2014 / Published: 25 November 2014
(This article belongs to the Special Issue Single Molecule Techniques)
View Full-Text   |   Download PDF [1981 KB, uploaded 26 November 2014]   |  

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. View Full-Text
Keywords: smFRET; lifetime FRET measurements; dye-linker dynamics; protein dynamics smFRET; lifetime FRET measurements; dye-linker dynamics; protein dynamics
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Höfig, H.; Gabba, M.; Poblete, S.; Kempe, D.; Fitter, J. Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV) Algorithm. Molecules 2014, 19, 19269-19291.

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