Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and differential scanning calorimetry study

Abstract

We present a combined theoretical (molecular dynamics, MD) and experimental (differential scanning calorimetry, DSC) study of the effect of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl chain-labeled fluorescent phospholipid analogs (C6-NBD-PC and C12-NBD-PC) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. DSC measurements reveal that <1 mol% of NBD-PC causes elimination of the pre-transition and a large loss of cooperativity of the main transition of DPPC. Labeling with C6-NBD-PC or C12-NBD-PC shifts the main transition temperature to lower or higher values, respectively. Following our recent report on the location and dynamics of these probes (BBA 1768 (2007) 467-478) in fluid phase DPPC, we present a detailed analysis of 100-ns MD simulations of systems containing either C6-NBD-PC or C12-NBD-PC, focused on their influence on several properties of the host bilayer. Whereas most monitored parameters are not severely affected for 1.6 mol% of probe, for the higher concentration studied (6.2 mol%) important differences are evident. In agreement with published reports, we observed that the average area per phospholipid molecule increases, whereas DPPC acyl chain order parameters decrease. Moreover, we predict that incorporation of NBD-PC should increase the electrostatic potential across the bilayer and, especially for C12-NBD-PC, slow lateral diffusion of DPPC molecules and rotational mobility of DPPC acyl chains.

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

“…In previous reports, we showed that incorporation of NBD-PC 14 and NBD-C n 33 causes an increase in the electrostatic potential difference between the bilayer and water phases. NBD-diC n PE presents two differences compared to these probes: the different configuration of the NBD fluorophore and the P-N dipole (the latter compared only to NBD-PC, as this dipole is absent in NBD-C n ); and their ionized state (whereas NBD-PC and NBD-C n were globally uncharged, NBD-diC n PE is anionic at neutral pH, and electroneutrality is maintained by the addition of sodium ions).…”

“…This behaviour agrees with experimental data obtained previously by our group. We measured the lipid/water partition coefficient of NBD-diC 14 PE into POPC bilayers, K p = 1.0 Â 10 6 . 17 For a related homologous series (fatty amines NBD-C n ), we obtained a linear variation of partition free energy with chain length, with slope DDG = À(2.4 AE 0.2) kJ mol À1 .…”

“…This inverted head group configuration is not seen in acyl chain NBD-labelled phospholipids C6-NBD-PC and C12-NBD-PC, for which no discernible differences in P-N angular distributions were observed compared to pure PC. 14 Mass density profiles across the bilayer normal direction of POPC, solvent, sodium ion (taken from the NBD-diC 18 PE simulation) and selected NBD-diC n PE derivatives. The probe and Na + curves were multiplied by 32 and 100 (respectively) for better visualisation.…”

Recent Developments in the Study of the Behavior of Fluorescent Membrane Probes in Lipid Bilayers: Molecular Dynamics Approach

“…1C), in fluid DPPC bilayers (0:64, 1:63, 4:60 NBD-PC/DPPC bilayers, T=323 K, t=100 ns) have been reported in two complementary studies. The first one addressed primarily the locations and dynamics of the probes (Loura & Prates Ramalho, 2007) whereas the second focused on their effects on the host bilayer properties (Loura et al, 2008). Parameterization of the NBD moiety required energy minimization for geometry and ab initio quantum mechanical calculations for partial atomic charges.…”

“…Parameterization of the NBD moiety required energy minimization for geometry and ab initio quantum mechanical calculations for partial atomic charges. Simulations were complemented with time-resolved fluorescence anisotropy (Loura & Prates Ramalho, 2007) and DSC (Loura et al, 2008) measurements, which provided experimental information on probe rotational dynamics and perturbation of the thermotropic behavior of DPPC bilayers, respectively. In these two studies it was observed that NBD fluorophores loop in the direction of the interface.…”