Diffusion-Enhanced FörsterResonance EnergyTransfer and the Effects of External Quenchers and the Donor QuantumYield.

The structural and dynamic properties of a flexible peptidicchaincodetermine its biological activity. These properties are imprintedin intrachain site-to-site distances as well as in diffusion coefficientsof mutual site-to-site motion. Both distance distribution and diffusiondetermine the extent of Förster resonance energy transfer (FRET)between two chain sites labeled with a FRET donor and acceptor. Bothcould be obtained from time-resolved FRET measurements if their individualcontributions to the FRET efficiency could be systematically varied.Because the FRET diffusion enhancement (FDE) depends on the donor-fluorescencelifetime, it has been proposed that the FDE can be reduced by shorteningthe donor lifetime through an external quencher. Benefiting from thehigh diffusion sensitivity of short-distance FRET, we tested thisconcept experimentally on a (Gly–Ser)6segment labeledwith the donor/acceptor pair naphthylalanine/2,3-diazabicyclo[2.2.2]oct-2-ene(NAla/Dbo). Surprisingly, the very effective quencher potassium iodide(KI) had no effect at all on the average donor–acceptor distance,although the donor lifetime was shortened from ca. 36 ns in the absenceof KI to ca. 3 ns in the presence of 30 mM KI. We show that the proposedapproach had to fail because it is not the experimentally observedbut the radiative donor lifetime that controls the FDE. Because ofthat, any FRET ensemble measurement can easily underestimate diffusionand might be misleading even if it employs the Haas–Steinbergdiffusion equation (HSE). An extension of traditional FRET analysisallowed us to evaluate HSE simulations and to corroborate as wellas generalize the experimental results. We demonstrate that diffusion-enhancedFRET depends on the radiative donor lifetime as it depends on thediffusion coefficient, a useful symmetry that can directly be appliedto distinguish dynamic and structural effects of viscous cosolventson the polymer chain. We demonstrate that the effective FRET rateand the recovered donor–acceptor distance depend on the quantumyield, most strongly in the absence of diffusion, which has to beaccounted for in the interpretation of distance trends monitored byFRET. [ABSTRACT FROM AUTHOR]