Lateral Distribution of NBD-PC Fluorescent Lipid Analogs in Membranes Probed by Molecular Dynamics-Assisted Analysis of Förster Resonance Energy Transfer (FRET) and Fluorescence Quenching

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.

Interaction of NBD-labelled fatty amines with liquid-ordered membranes: a combined molecular dynamics simulation and fluorescence spectroscopy study

“…As illustrated in Fig. 12 32 This agrees with the essentially identical fluorophore locations for the three probes, 28,32 as well as with the similarity in their average fluorescence lifetimes in Chol-free bilayers (B6 ns being reported for both C 6 -NBD-PC in fluid DPPC 94 and NBD-PE in POPC 95 ). Fig.…”

Behaviour of NBD-head group labelled phosphatidylethanolamines in POPC bilayers: a molecular dynamics study

“…Whereas the decay of N-propylamino NBD (NBD-C 3 ) emission may be described by a single exponential function in a variety of pure solvents, 82 the huge possible variations in environment polarity that may be experienced by membrane-inserted fluorophores of NBD-PC and NBD-diC n PE dictated otherwise. For example, in fluid DPPC vesicles (50 1C) with 0.1 mol% NBD lipid, both NBD-diC 16 PE (t 1 = 0.87 ns (25% pre-exponential), t 2 = 6.61 ns (75%), intensity-weighted average lifetime hti = 6.37 ns 20 ) and C6-NBD-PC (t 1 = 0.94 ns (20%), t 2 = 6.16 ns (80%), hti = 5.97 ns 27 ) present bi-exponential decays, with similar contributions of a short lifetime component, close to that measured for NBD-C 3 in water (1.0 ns). 82 Similarly heterogeneous decays were measured in other lipid systems.…”

“…[17][18][19] Used as FRET donors, NBD-diC n PE probes form, together with acceptor rhodamine B PE derivatives, a very well-known Förster pair for the studies of membrane organization [20][21][22][23][24] as well as quantification of lipid mixing and exchange. 25 Alternatively, NBD probes can be used as acceptors to chromophores including diphenylhexatriene 26,27 or trans-parinaric acid, 28 or even as donors and acceptors simultaneously in homo-FRET studies. 20 Several fluorescence spectroscopic and microscopic studies have also indicated that, contrary to the vast majority of membrane fluorescent probes, doubly saturated long-chained NBD-diC n PE partitions favourably to liquid ordered (lo) phases in systems displaying lo/liquid disordered phase coexistence, 22,24,29,30 although the phase preference depends both on the probe acyl chain length and the composition of the underlying lipid mixture.…”

Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching

“…Lipids and cholesterol analogues labeled with an NBD fluorophore have also been used to detect the presence of lipid domains in cell membranes (Stubbs et al 1989). NBD-labeled phospholipids have often been applied to determine the depth position of other fluorophores within membranes by FRET (Feigenson 1997); (Posokhov and Ladokhin 2006); (Loura 2012). Despite the broad application range of NBD-PE probe, a limited number of studies have attempted to characterize its structure in the membrane environment and the immersion depth of the NBD groups (Mazères et al 1996).…”