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

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, sigma, 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 sigma 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 appear 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 kappa2 can be calculated for various mutual dye orientations. The resulting kappa2 distribution is very much narrower than the limiting values of 0 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 sigma 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.

journal_name

Biopolymers

journal_title

Biopolymers

authors

Parkhurst LJ,Parkhurst KM,Powell R,Wu J,Williams S

doi

10.1002/bip.10138

subject

Has Abstract

pub_date

2001-01-01 00:00:00

pages

180-200

issue

3

eissn

0006-3525

issn

1097-0282

journal_volume

61

pub_type

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