Extended Förster theory for determining intraprotein distances: 2. An accurate analysis of fluorescence depolarisation experiments

The extended Förster theory (EFT) is for the first time applied to the quantitative determination of the intramolecular distances in proteins. It is shown how the EFT (J. Chem. Phys., 1996, 105, 10896) can be adapted to the analyses of fluorescence depolarisation experiments based on the time-correlated single photon counting technique (TCSPC). The protein system studied was the latent form of plasminogen activator inhibitor type I (PAI-1), which was mutated and labelled by the thiol reactive BODIPY® derivative {N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide}. The energy migration occurs within pairs of photophysically identical donor groups that undergo reorientational motions on the timescales of energy migration and fluorescence relaxation. Unlike all models currently used for analysing fluorescence TCSPC data, the EFT explicitly accounts for the time-dependent reorientations that influence the rate of electronic energy transfer/migration in a complex manner. The complexity is related to the “κ² problem”, which has been discussed for years. The EFT brings the analyses of DDEM data to the same level of molecular description as in ESR and NMR spectroscopy, i.e. it yields microscopic information about the reorientation correlation times, the order parameters, as well as inter-chromophoric distances.