Fluorescence Studies of Complex Systems: Organisation of Biomolecules

The homo and hetero dimerisation of two spectroscopically different chromophores were studied, namely: 4,4difluoro-4-bora-3a,4a-diazas-indacene (g-BODIPY) and its 5-styryl-derivative (r-BODIPY). Various spectroscopic properties of the r-BODIPY in different common solvents were determined. It was shown that gand r-BODIPY in the ground state can form homoas well as hetero dimers. We demonstrate that the ganglioside GM1 in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, which is an argument in favour of self-aggregation of GM1 being an intrinsic property of the GM1. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides. An algorithm is presented that quantitatively accounts for donor–donor energy migration (DDEM) among fluorophorelabelled proteins forming regular non-covalent polymers. The DDEM algorithm is based on Monte Carlo (MC) and Brownian dynamics (BD) simulations and applies to the calculation of fluorescence depolarisation data, such as the fluorescence anisotropy. Thereby local orientations, as well as reorienting motions of the fluorescent groups are considered in the absence and presence of DDEM among them. A new method, in which a genetic algorithm (GA) was combined with BD and MC simulations, was developed to analyse fluorescence depolarisation data collected by the time-correlated single photon counting technique. It was applied to study g-BODIPY-labelled filamentous actin (F-actin). The technique registered the local order and reorienting motions of the fluorophores, which were covalently coupled to cysteine 374 (C374) in actin and interacted by means of electronic energy migration within the polymer. Analyses of F-actin samples composed of different fractions of labelled actin molecules revealed the known helical organiszation of F-actin, and demonstrated the usefulness of this technique for structure determination of complex protein polymers. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed position of C374 at a high filament radius. In addition, polymerisation experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.