Extended Förster theory of donor–donor energy migration in bifluorophoric macromolecules. Part II. Method for determining intramolecular distances with experimental validation using mono and bifluorophoric systems

Abstract

Recently an approximate theory was presented and applied for determining intramolecular distances in proteins. The rate of donor–donor energy migration (DDEM) is extracted and analysed from fluorescence depolarisation experiments by means of the DDEM model (Karolin et al., Biophys. J., 1998, 74, 11; Bergström et al., Proc. Natl. Acad. Sci., 1999, 96, 12477). Previously an extended Förster theory (EFT) was derived (Johansson et al., J. Chem. Phys., 1996, 105, 10896), which accounts for DDEM between reorienting molecules. For the first time, this rigorous theory is applied for analysing time-resolved fluorescence depolarisation data, accumulated by using the time-correlated single photon counting (TCSPC) technique. A simulation–deconvolution algorithm is presented which reduces the need of the DDEM model (Edman et al., Phys. Chem. Chem. Phys., 2000, 2, 1789), and other approximate theories (Edman et al., Mol. Phys., submitted). Two bifluorophoric systems were studied, namely; 1,32-dihydroxy-dotriacontane-bis(rhodamine) 101 ester solubilised in lipid vesicles, and bis(9-anthrylmethyl-phosphonate) bisteroid dissolved in propane-1,2-diol. The bis-rhodamine molecules span across lipid bilayers, so that the two rhodamine moieties of the molecule are localised on opposite sides of a bilayer. From the analyses of the fluorescence anisotropy, the donor–donor distances were determined to be 36.5±1 and 21.0±1.5 Å, for the membrane spanning molecule and the bisteroid, respectively. The results are in good agreement with independent studies.