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

Abstract

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.-Å. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1087–1097) 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-dimethyl-4-bora-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 poly-fluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.