Abstract
Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed.
Highlights
Because of its strong intermolecular distance dependence, Förster resonance energy transfer (FRET) has multiple applications in membrane biophysics, such as membrane protein mapping, lateral heterogeneity, determination of the transverse location of fluorescent residues/labels inside the membrane, protein/lipid selectivity, and membrane protein oligomerization [1], both in spectroscopic studies and, more recently, under the microscope [2]
At long times the decay curve becomes asymptotically parallel to that in the absence of acceptor, as the FRET rate becomes negligible relative to the intrinsic decay rate
Eventual deviations from the theoretical decay law assuming uniform probe distribution could be ascribed to the acceptors. This probe and host lipid setting was chosen given that detailed atomic-level topological and orientation information was available from molecular dynamics (MD) simulation studies [10,11,12,13,14], which could be used as input in the FRET analysis
Summary
Because of its strong intermolecular distance dependence (and sensitivity to spatial distribution), Förster resonance energy transfer (FRET) has multiple applications in membrane biophysics, such as membrane protein mapping, lateral heterogeneity (membrane domains), determination of the transverse location (depth) of fluorescent residues/labels inside the membrane, protein/lipid selectivity (preference of a specific lipid for the protein vicinity), and membrane protein oligomerization [1], both in spectroscopic studies and, more recently, under the microscope [2]. Even though FRET is still commonly used as a qualitative indicator of chromophore proximity without accounting for its actual kinetics, full quantitative exploitation of its potential involves modeling of the FRET observables To this effect, the decay of donor fluorescence in the presence of an acceptor is especially useful. One of the simplest possible scenarios is the analysis of FRET data with the formalism for uniform planar distribution of fluorophores in a monophasic bilayer. In this case, the donor fluorescence decay (iDA(t)) is given by: t iDA ( t ) exp exp R02 c
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