Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics...

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Bibliographic Details
Published in:Nature communications 2015-04, Vol.6 (1), p.6992-6992, Article 6992
Main Authors: Kim, Jae-Yeol, Kim, Cheolhee, Lee, Nam Ki
Format: Article
Language:English
Subjects:
140/125
631/1647
631/1647/527/2047
631/45/612/1229
631/57/2272
Biomolecules
Deinococcus
Diffusion
DNA, Single-Stranded - metabolism
DNA-Binding Proteins - metabolism
Dynamics
E coli
Energy transfer
Escherichia coli
Fluorescence
Fluorescence Resonance Energy Transfer - methods
Humanities and Social Sciences
Liposomes
Markov chains
multidisciplinary
Nanotechnology - methods
Photons
Proteins
Real time
Science
Science (multidisciplinary)
Temporal resolution
Tethering
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Summary:Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s –1 between two conformers of a Holliday junction. The rapid diffusional migration of Deinococcus radiodurans single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of Escherichia coli SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution. Single-molecule fluorescence resonance energy transfer is widely used to probe biomolecular dynamics, but is limited by its temporal resolution. Here, Kim et al . push the limit to submillisecond for the duration of tens of milliseconds by tethering target molecules to liposomes in buffer solutions.
ISSN:2041-1723
2041-1723
DOI:10.1038/ncomms7992