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We used time-dependent fluorescence energy transfer of externally quenched and nonquenched samples, and global analysis of the data, to recover the end-to-end distance distributions and diffusion coefficients of flexible fluorescent molecules in low-viscosity solution. The fluorescence decays of tryptamine covalently linked to a dansyl acceptor by a polyethylene chain of 22 methylene groups were measured by the frequency-domain method. The data were fitted using numerical solutions of the diffusion equation which predicts the time- and distance-dependent population of the excited-state donors in the presence of energy transfer, followed by transformation to the frequency domain for nonlinear least-squares fitting to the experimental data. From the simulation study we found that the time- and distance-dependent population of the excited-state donors are significantly different for nonquenched and quenched samples and that the effects of end-to-end diffusion on the donor decay is decreased by collisional quenching. Importantly, the resolution is dramatically improved by the use of simultaneous analysis of quenched and nonquenched samples. This method was applied to the tryptamine-dansyl system using acrylamide as an external quencher. The recovered initial ( t = 0) distance distribution, R  av = 18.9 Å, hw = 17.1 Å, is very similar to that obtained for diffusion-free conditions. The end-to-end diffusion coefficient of D = 1.26 × 10 -5 cm 2 /s is comparable to that expected for molecules the size of indole and dansyl. This value is about twice smaller than that obtained from diffusion-dependent intermolecular energy transfer using unlinked indole and dansylamide as the donor and acceptor, respectively, which may reflect the effects of the linker on diffusion of the chromophores.

journal of physical chemistry

End-to-end diffusion and distance distributions of flexible donor-acceptor systems observed by intramolecular energy transfer and frequency-domain fluorometry; enhanced resolution by global analysis of externally quenched and nonquenched samples