Measuring conformational dynamics: A new FCS-FRET approach

Tedman Torres, Marcia Levitus

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

Fluorescence correlation spectroscopy (PCS) has recently emerged as a powerful technique to study conformational dynamics of biomolecules, but often its applicability is limited by the difficulty of separating the contributions of kinetics from those due to diffusion. We present a new approach based on the simultaneous analysis of the auto- and cross-correlation functions of the intensities measured in two independent detectors for a donor - acceptor labeled biomolecule. Fluctuations in fluorescence intensity are a consequence of diffusion and the variations in fluorescence resonance energy transfer (FRET) efficiency due to changes in donor -acceptor distance. Although the complete description of the correlation functions requires that diffusion is well-characterized, the ratio of any two correlation functions depends on kinetic parameters only. This provides a means by which kinetic information can be obtained independently of the diffusion contributions. As proof of principle, we reanalyze data obtained in previous work with nucleosomes. In contrast to our previous work, where a donor-only sample was used to characterize diffusion, we now show that the same kinetic information can be obtained from a single experiment with a double-labeled biomolecule. This eliminates not only the need of a reference, but also artifacts associated with changes in the observation volume between measurements.

Original languageEnglish (US)
Pages (from-to)7392-7400
Number of pages9
JournalJournal of Physical Chemistry B
Volume111
Issue number25
DOIs
StatePublished - Jun 28 2007

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resonance fluorescence
energy transfer
Biomolecules
kinetics
Kinetics
Fluorescence
Volume measurement
fluorescence
Nucleosomes
Autocorrelation
Kinetic parameters
cross correlation
autocorrelation
artifacts
Fluorescence Resonance Energy Transfer
Spectroscopy
Detectors
detectors
spectroscopy
Experiments

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Measuring conformational dynamics : A new FCS-FRET approach. / Torres, Tedman; Levitus, Marcia.

In: Journal of Physical Chemistry B, Vol. 111, No. 25, 28.06.2007, p. 7392-7400.

Research output: Contribution to journalArticle

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