Computer Aided Distance Reconstruction in Single Molecule FRET Experiments through Combination with Dye Dynamics from Molecular Dynamics Simulations

Abstract

Fluorescence resonance energy transfer (FRET) experiments probe molecular distances via the efficiency of energy transfer from an excited donor dye to its acceptor counterpart. To study flexibilities and structural changes in biomolecules, single molecule FRET settings allow probing distance distributions and fluctuations.However, the measured energy transfer efficiency depends not only on the distance between the two dyes, but also on their mutual orientation which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions have to be made such as an isotropic orientation distribution. These approximations severely limit the accuracy of the distance distributions extracted from FRET experiments alone.Here we show that estimates of the distance distributions can be improved considerably by combining measured efficiency distributions with dye orientation statistics obtained from molecular dynamics (MD) simulations. Quantitative agreement of calculated efficiency distributions from simulations with the experimental efficiency distributions was obtained for poly-proline chains with Alexa488 and Alexa594 dyes attached. The experimentally observed heterogeneity was related to the presence of cis-isomers and previously undetected distinct dye conformers.This agreement of our in silico FRET approach with experiments provides a solid basis to use the obtained dye orientation dynamics from MD simulations in the distance reconstruction. We propose a transfer-function based approach and show that the poly-proline all-trans peak location in the reconstructed distances deviates by only 0.35A from the true peak location in contrast to a 6.2A deviation when employing the isotropic approximation of the dye orientation.