Conformational changes in complex macromolecules studied by single donor–acceptor pair fluorescence

Abstract

A single donor–acceptor (D–A) pair with a fluctuating FRET rate F is studied theoretically in the frame of a “two-state model” in which the FRET rate F fluctuates taking two values, F₁ and F₂, with average rates B for the forward and b for the backward transitions. Theoretical expressions are derived for the autocorrelation function g⁽²⁾_DA(τ) and for the Mandel parameter Q^D,A(τ) allowing for background emission. Fluctuating intensities I_D,A(t) and FRET efficiency E(t) = I_A(t)/[I_A(t) + I_D(t)] are calculated with the help of the Monte Carlo technique. The probability w^D,A_N(T) of finding N photons in a time interval T, and the distribution of the FRET efficiency P(E) are found by statistical treating of the fluctuating intensities I_D,A(t). The shape of the distribution w^D,A_N(T) enables one to find the values of the parameters: F₁, F₂, and b/B. The influence of the background light on g⁽²⁾_DA(τ), Q^D,A(τ) and w^D,A_N(T) is studied. It is shown how the background light influences the ratio b/B found from the analysis of w^D,A_N(T) and P(E).