Macromolecular Dynamics Observed by Intramolecular Energy Transfer Using Frequency-Domain Fluorometry

Abstract

We used time-dependent fluorescence energy transfer to recover the site-to-site distance distributions and diffusion coefficients for a flexible donor-acceptor pair and for the proteins melittin (random coil and α-helix states) and troponin I (native and denatured). Energy transfer occurred from single tryptophan residues to dansyl acceptors. The differential equation describing the donor population in the presence of site-to-site diffusion (Biopolymers 17:11-31, 1978) was solved numerically. These data were used to obtain the predicted frequency-response for least squares analysis. Global analysis of progressively quenched samples was used to enhance the resolution of recovered parameters. The validity of the recovered distance distributions and diffusion coefficients was verified by simulations and by studies of the flexible methylene-chain linked donor-acceptor pair in solutions of varied viscosity.