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

Abstract

Publisher Summary Parameters that define a distance distribution for Forster resonance energy transfer (FRET) donor and acceptor pairs can be extracted from time-resolved FRET measurements. The ultimate interest in extracting such parameters is in determining distances that are relatively static on a nonosecond time scale. In turn, these distances provide information about multiple conformations of macromolecules and about the kinetics and energetics that link such conformations. Not only the mean distance, but parameters from higher moments of the distribution in particular the width— provide the information about the flexibility of macromolecules. These distances and particularly the changes in distances can be determined to high precision allowing FRET to play a key role in providing highly precise distances in solution. This chapter reviews a few essentials of FRET with attention to experimental and computational methods and shows the relationship of distance parameters to macromolecular changes in the field of DNA and DNA–TATA-binding protein (TBP) interactions. These structural changes in turn are intimately linked to various thermodynamic properties. 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 intra-molecular distances in solution. The width of the P(R) distribution, σ, 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.