Distance Parameters Derived from Time-Resolved Förster Resonance Energy Transfer Measurements and Their Use in Structural Interpretations of Thermodynamic Quantities Associated with Protein-DNA Interactions

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Abstract

Parameters that characterize a distance distribution P(R) can be obtained from time-resolved FRET measurements. These measurements can involve various combinations of donor-detected FRET and acceptor-detected FRET constrained by steady state emission intensity differences between the donor and that of the donor in the presence of an acceptor. Highly precise average interdye distances R can ultimately lead to precise intramolecular distances in solution. The width of the P(R) distribution, δ, preferably and more precisely after removal of the tether contributions, yields a measure of conformational equilibria and of conformational dynamics of the macromolecule to which the probes are attached. FRET measurements combined with equilibrium determinations and with rapid-mixing or relaxation kinetics provide structure-energy, entropy profiles of intermediates and transition states along the reaction coordinate.

Original languageEnglish (US)
Pages (from-to)235-262
Number of pages28
JournalMethods in enzymology
Volume379
DOIs
StatePublished - 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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