Resolution of end‐to‐end diffusion coefficients and distance distributions of flexible molecules using fluorescent donor‐acceptor and donor‐quencher pairs

We used time‐dependent fluorescence energy transfer, time–dependent collisional quenching, and global analysis of the data resulting from these through–space and contact interactions to recover the end–to–end distance distributions and diffusion coefficients of flexible fluorescent molecules. The fluorescence decays of covalently linked tryptamine–acceptor and tryptamine–quencher pairs were measured by the frequency–domain method. These data were fit using numerical solutions of the differential equation, which predicts the time–and distance–dependent population of the excited state donors in the presence of energy transfer or collisional quenching, followed by transformation to the frequency domain for nonlinear least–squares comparison with the experimental data. We found that the energy transfer data for the donor–acceptor pair alone were adequate to recover the starting distribution and the end–to–end diffusion coefficient; however, the resolution is dramatically improved by the use of both the through–space and contact interactions.