On the breakdown of Förster energy transfer theory due to solvent effects: atomistic simulations unveil...

Förster resonance energy transfer (FRET) is a powerful technique used to investigate the conformational preferences of biosystems, and molecular simulations have emerged as an ideal complement to FRET due to their ability to provide structural models that can be compared with experiments. This synergy is however hampered by the approximations underlying Förster theory regarding the electronic coupling between the participating dyes: a dipole-dipole term attenuated by a simple dielectric screening factor 1/n ² that depends on the refractive index of the medium. Whereas the limits of the dipole approximation are well-known, detailed insights on how environment effects deviate from the 1/n ² assumption and modify the R ^-6 distance dependence that characterizes FRET as a spectroscopic ruler are still not well understood, especially in biosystems characterized by significant structural disorder. Here we address this using a rigorous theoretical framework based on electrostatic potential-fitted transition charges coupled to an atomistic polarizable classical environment, which allows investigation of dielectric screening in atomic detail in extended simulations of disordered systems. We apply this strategy to investigate the conformational preferences of calmodulin, a protein that plays a major role in the transmission of calcium signals. Our results indicate that dielectric screening displays an exponential decay at donor/acceptor separations below 20 Å, significantly modifying the R ^-6 distance dependence widely adopted in FRET studies. Screening appears to be maximized at separations ∼15 Å, a situation in which the fluorophores are partially excluded from the solvent and thus screening is dictated by the more polarizable protein environment.