On determining intramolecular distances from donor-donor energy migration (DDEM) within bifluorophoric macromolecules

Recently a model based on donor-donor energy migration (DDEM) was developed for examining structure-function properties of biomacromolecules such as proteins (J. Chem. Soc., Faraday Trans., 1996, 92, 1563). Unlike the extended Forster theory (EFT; J. Chem. Phys., 1996, 105, 10896) the DDEM model is straightforward to apply in the analyses of fluorescence depolarisation experiments, as obtained by the time-correlated single photon counting (TCSPC) technique. In order to test the validity of the DDEM model, the EFT was used to create synthetic depolarisation data. These mimic true TCSPC experiments and cover a wide range of physical conditions, which are difficult to arrange for in real experiments with model systems. In particular, the relative rate of DDEM (omega) and the rotation correlation time (phi) of the donor molecules was examined. The DDEM rates obtained from the analyses were compared to the true rates. From these results the relative error of the intramolecular distances were calculated. For values 1 < 12 omega phi < 25, the DDEM model is slightly overestimating the distances. Typically, the distances determined with the DDEM model are overestimated by 5-10%.