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Fluorescence Lifetime Tuning—A Novel Approach to Study Flip-Flop Kinetics in Supported Phospholipid Bilayers

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Abstract

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.

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Acknowledgements

We thank Dr. Alexander Deyneka (Institute of Physics, ASCR, Czech Republic) for modifications and characterization of the ITO supports, Dr. Andrey S. Klymchenko, (Université de Strasbourg, Laboratoire Biophotonique et Pharmacologie, Illkirch, Cedex, France) for the synthesis of the F2N12S probe and Prof. Dr. Jörg Enderlein (Universität Göttingen, Germany) for providing us with the source code for theoretical calculations of dipole-interface interactions. We would like to acknowledge the Grant Agency of the Academy of Sciences of the Czech Republic (M.H. and P.J. via MEM/09/E006) and the Ministry of Education, Youth and Sports of the Czech Republic (A.K., A.B. and J.S. via LC06063) for financial support.

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Authors and Affiliations

  1. J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v. v. i., Dolejskova 3, 18223, Prague 8, Czech Republic

    Anna Kułakowska, Piotr Jurkiewicz, Jan Sýkora, Aleš Benda & Martin Hof

  2. Laboratoire Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401, Illkirch, Cedex, France

    Yves Mely

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  1. Anna Kułakowska
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  2. Piotr Jurkiewicz
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  3. Jan Sýkora
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  4. Aleš Benda
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  5. Yves Mely
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  6. Martin Hof
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Correspondence to Martin Hof.

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Kułakowska, A., Jurkiewicz, P., Sýkora, J. et al. Fluorescence Lifetime Tuning—A Novel Approach to Study Flip-Flop Kinetics in Supported Phospholipid Bilayers. J Fluoresc 20, 563–569 (2010). https://doi.org/10.1007/s10895-009-0581-9

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  • DOI: https://doi.org/10.1007/s10895-009-0581-9

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