Partial donor-donor energy migration (PDDEM):: A novel fluorescence method for internal protein distance measurements

We show that the photophysics of chemically identical but photophysically non-identical fluorescent pairs can be used for measuring distances within proteins. For this purpose, the theory of partial donor-donor energy migration (PDDEM, S. Kalinin, J. G. Molotkovsky and L. B.-Angstrom. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1057-1097) was applied for distance measurements between BODIPY groups covalently linked to cystein residues in plasminogen activator inhibitor of type 2 (PAI-2). Two sulfhydryl specific derivatives of BODIPY were used namely: N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide and N-(4.4-difluoro-5.7-ditriethyl-4-bona-3a,4a-diaza-s-indacene-3-yl) methyl iodoacetamide. To determine distances, the time-resolved fluorescence relaxation for two singly labelled forms of PAI-2, as well as the corresponding doubly labelled protein were combined and analysed in a global manner. Fluorescence depolarisation experiments on the labelled mutants were also analysed. The distances determined by PDDEM were in good agreement to those obtained from donor-donor energy migration (DDEM) experiments and structural data on PAI-2. The PDDEM approach allows for the use of very different fluorescent probes, which enables wide range of distances to be measured. The PDDEM model also provides a rational explanation to why previous observations of polyfluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.