Joint refinement of FRET measurements using spectroscopic and computational tools

The variability of the orientation factor is a long-standing challenge in converting FRET efficiency measurements into donor-acceptor distances. We propose the use of molecular dynamics (MD) simulations to characterize orientation distributions and thus improve the accuracy of distance measurements. Here, we test this approach by comparing experimental and simulated FRET efficiencies for a model donor-acceptor pair of enhanced cyan and enhanced yellow FPs connected by a flexible linker. Several spectroscopic techniques were used to characterize FRET in solution. In addition, a series of atomistic MD simulations of a total length of 1.5 μs were carried out to calculate the distances and the orientation factor in the FRET-pair. The resulting MD-based and experimentally measured FRET efficiency histograms coincided with each other, allowing for direct comparison of distance distributions. Despite the fact that the calculated average orientation factor was close to 2/3, the application of the average κ² to the entire histogram of FRET efficiencies resulted in a substantial artificial broadening of the calculated distribution of apparent donor-acceptor distances. By combining single pair-FRET measurements with computational tools, we demonstrate that accounting for the donor and acceptor orientation heterogeneity is critical for accurate representation of the donor-acceptor distance distribution from FRET measurements.