Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes

Lawrence J. Parkhurst, Kay M. Parkhurst, Robyn Powell, Jiong Wu, Sarah Williams

Research output: Contribution to journalReview article

48 Scopus citations

Abstract

Time-resolved Förster resonance energy transfer (trFRET) has been used to obtain interdye distance distributions. These distributions give the most probable distance as well as a parameter, σ, that characterize the width of the distribution. This latter parameter contains information not only on the flexibility of the dyes tethered to macromolecules, but on the flexibility of the macromolecules. Both the most probable interdye distance as well as σ provide insight into DNA static bending and DNA flexibility. Time-resolved fluorescence anisotropy and static anisotropy measurements can be combined to provide a measure of the cone angle within which the tethered dyes appeal to wobble. When this motion is an order of magnitude faster than the average lifetime that characterizes transfer, an average value of the dipolar orientational parameter K2 can be calculated for various mutual dye orientations. The resulting K2 distribution is very much narrower than the limiting values of O and 4, allowing more precise distances and distance changes to be determined. Static and time-resolved fluorescence data can be combined to constrain the analyses of DNA-protein kinetics to provide thermodynamic parameters for binding and for conformational changes along a reaction coordinate. The parameter σ can be used to model multiple DNA-protein complexes with varying DNA bend angles in a global fitting of trFRET data. Such a global fitting approach has shown how the range of bends in single base DNA variants, when bound by the TATA binding protein (TBP), can be understood in terms of two limiting forms. Time-resolved FRET, combined with steady-state FRET, can be used to show not only how osmolytes affect the binding of DNA to proteins, but also how DNA bending depends on osmolyte concentration in the DNA-protein complexes.

Original languageEnglish (US)
Pages (from-to)180-200
Number of pages21
JournalBiopolymers
Volume61
Issue number3
DOIs
StatePublished - Jan 1 2001

Keywords

  • DNA bending
  • DNA-protein complexes
  • DNA-protein kinetics
  • Energy transfer
  • Fluorescence anisotropy
  • Fluoresence
  • Förster resonance energy transfer

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

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