Effect of Diffusion on Resonance Energy Transfer Rate Distributions: Implications for Distance Measurements.

Intrinsically disordered protein regions and many other biopolymers lack the three-dimensional structure that could be determined by X-ray crystallography or NMR, which encourages the application of alternative experimental methods. Time-resolved resonance energy transfer data are often used to measure distances between two fluorophores attached to a flexible biopolymer. This is complicated by the rotational and translational diffusion of the fluorophores and by nonmonoexponential donor decay in the absence of the acceptor. Equation IDA(t) = ID(t)·F(t) is derived here, which is applicable regardless of whether ID(t) is monoexponential. ID(t) and IDA(t) are the δ-excitation donor emission decays in the absence and in the presence of the acceptor; F(t) contains information about energy transfer, donor–acceptor distance distribution, and diffusion dynamics. It is shown that in the absence of rotational and translational diffusion, F(t) is a continuous distribution of exponentials, whereas in the presence of ...