Fluorescence depolarization studies of filamentous actin analyzed with a genetic algorithm |
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Authors: | Marushchak Denys Grenklo Staffan Johansson Thomas Karlsson Roger Johansson Lennart B-A |
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Institution: | *Department of Chemistry; Biophysical Chemistry, Umeå University, Umeå, Sweden; and ?Department of Cell Biology, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden |
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Abstract: | A new method, in which a genetic algorithm was combined with Brownian dynamics and Monte Carlo simulations, was developed to analyze fluorescence depolarization data collected by the time-correlated single photon-counting technique. It was applied to studies of BODIPY-labeled 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 electronic energy migration within the actin polymers. Analyses of F-actin samples composed of different fractions of labeled actin molecules revealed the known helical organization of F-actin, demonstrating 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, polymerization experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin. |
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Keywords: | C∞ rotational axis of the helical structure D donor of electronic energy D" target="_blank">D director frame of the donor D(t) the observed fluorescence difference curve f mole fraction of labeling R0 the Förster radius r(t) the time-resolved fluorescence anisotropy Sj second-rank order parameter of the jth fluorophore TCSPC time-correlated single-photon counting χ2 the statistical test parameter |
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