End-to-end diffusion coefficients and distance distributions from fluorescence energy transfer measurements: enhanced resolution by using multiple acceptors with different Förster distances.

We measured distance distributions and end-to-end diffusion coefficients of donor-acceptor pairs linked by a flexible methylene chain using frequency-domain fluorescence energy transfer measurements. The donor was an indole group, and two acceptors with different Förster distances were used. The uncertainties in the recovered parameters describing the end-to-end distance distribution and diffusion coefficient were rather large when each donor-acceptor pair was analyzed separately. It was not possible to resolve distance distributions in the presence of intra-molecular diffusion when the Förster distance was comparable to the mean and half-width of the distribution. Global analysis using two acceptors dramatically improved the resolution. Surprisingly, the Förster distances need not be very different, and a 20% difference between the two R0 values resulted in substantial improvements in resolution. Both the simulations and the experiments suggest the benefit of using global analysis with different Förster distances to obtain reliable distance distribution parameters in the presence of diffusion.