Direct calculation of Förster orientation factor of membrane probes by molecular simulation

Fluorescence is a major tool in biophysics, in particular in membrane studies. However, because lipids are intrinsically nonfluorescent, fluorescence studies of membranes depend on the use of extrinsic probes, whose properties and behavior inside the bilayer are often poorly understood. One important parameter in fluorescence spectroscopy for which there is no experimental technique suited for its determination is the Förster resonance energy transfer (FRET) orientation factor, or κ2. Its theoretical value for the so-called dynamic isotropic limit is usually used (2/3), but this approximation is debatable because of the intrinsic anisotropic nature of bilayers. Atomistic molecular dynamics simulations were performed in this work in order to determine the distribution and average value of the FRET orientation factor for homotransfer between fluorescent membrane probes 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)-sn-glycero-3-phosphocholine (C6-NBD-PC) and 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) in a fluid bilayer of 1,2-dipalmitoyl-sn-3-glycerophoshocholine (DPPC, T = 323 K). Although the distributions generally resemble the analytical solutions for isotropic dipoles, the average value for C12-NBD-PC is ∼30% higher than the 2/3 dynamic isotropic limit value. This application illustrates the utility of the present methodology for obtaining accurate estimates of the FRET orientation factor.