On the Quantitative Analysis of Electronic Energy Transfer/migration in Proteins Studied by Fluorescence Spectroscopy

Two recently developed theories of electronic energy transfer/migration were for the first time applied to real protein systems for extracting molecular distances. The partial donor-donor energy migration (PDDEM) is an extension to the previously developed donor-donor energy migration (DDEM, F Bergström et al PNAS 96, 1999, 12477) which allows using chemically identical but photophysically different fluorophores in energy migration experiments. A method based on fluorescence quenching was investigated and applied to create an asymmetric energy migration between fluorophores which were covalently and specifically attached to plasminogen activator inhibitor type 2 (PAI-2). It was also shown experimentally that distance information can be obtained if the fluorescence relaxation for photophysically identical donors, exhibits multi-exponential relaxation. An extended Förster theory (EFT) that was previously derived (L. B.-Å. Johansson et al J. Chem. Phys., 1996, 105) ha been developed for analysis of donor-acceptor energy transfer systems as well as DDEM systems. Recently the EFT was also applied to determine intra molecular distances in the protein plasminogen activator inhibitor type 1 (PAI-1) which was labelled with a sulfhydryl specific derivative of BODIPY. The EFT explicitly accounts for the time-dependent reorientations which in a complex manner influence the rate of electronic energy transfer/migration. This difficulty is related to the “κ-problem”, which has been solved. It is also shown experimentally that the time-correlated single-photon counting (TCSPC) data is sensitive to the mutual configuration between the interacting fluorophores. To increase the accuracy in the extracted parameters it is furthermore suggested to collect the fluorescence data under various physico-chemical conditions. It was also shown that the Förster theory is only valid in the initial part of the fluorescence decay.