Figure 4: Assessing distributions of inter-dye distances with fluorescence lifetime information.

With pulsed interleaved excitation, it is possible to determine the relative donor lifetime in the presence of the acceptor, τ_DA/τ_D. This parameter provides information about the variance of the underlying distribution of transfer efficiencies. (left) In the case of a static distribution of distances in a 20-mer polyproline peptide (R. Best et al., 2007; Schuler et al., 2005), the values of 〈τ_DA〉/τ_D cluster close to the diagonal, which corresponds to a single fixed distance (static FRET line), 〈τ_DA〉/τ_D = 1 − ε. (right) However, for a broad and rapidly sampled distribution, for example the intrinsically disordered peptide sNh⁻, values of τ_DA/τ_D cluster above the diagonal. This vertical displacement provides a measure of the variance of the underlying distribution of transfer efficiencies, σ². In this way, lifetime vs. transfer efficiency plots can be used to assess the quality of polymer models (i.e., self-avoiding walk (Zheng et al., 2018), or worm-like chain (O’Brien, Morrison, Brooks, & Thirumalai, 2009)) commonly used to describe FRET-labeled biomolecules. The error of σ² was estimated assuming an uncertainty of ~0.1 ns for both τ_D and τ_DA.