Figure 9.

Fast translational diffusion of the donor and acceptor dyes affects the recovered apparent donor–acceptor distribution due to averaging during their fluorescence lifetime. Effects of dye diffusion on apparent DA distance distributions, x(R_app). (A) Apparent DA distance distributions, x(R_app) recovered from a fluorescence decay of a donor with a lifetime of 4 ns attached to amino acid F379C and an acceptor attached to amino acid D467C of the hGBP1 protein structure (PDB-ID: 1F5N) in dependence of the diffusion coefficients of the donor D_D and the acceptor D_A = ¹/₂D_D without interaction of the dyes (left) and with interaction of the dyes (right) with the protein surface. Interacting dyes close to the protein surface diffused 10 times slower. (B) Apparent distances of a two-state system in dynamic exchange. The equally populated discrete states R_DA⁽¹⁾ = 40 Å and R_DA⁽²⁾ = 60 Å (R₀ = 52 Å) are in dynamic exchange with a rate constant k_dyn. The resulting biexponential FRET-induced donor decay was converted to yield two apparent distances (orange lines). Using these apparent distances, the average distance (black) was calculated. The gray line is the static average of the two distances.