2H-NMR study and molecular dynamics simulation of the location, alignment, and mobility of pyrene in POPC bilayers

Abstract

The alignment of pyrene in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer was investigated using two different approaches, namely solid-state (2)H-NMR spectroscopy and molecular dynamics (MD) simulations. Quadrupolar splittings from (2)H-NMR spectra of deuterated pyrene-d(10) in an oriented lipid bilayer give information about the orientation of C-D bonds with respect to the membrane normal. From MD simulations, geometric information is accessible via trajectories. By defining molecular and bond order parameters, the data from MD trajectories and NMR spectra can be compared straightforwardly. To ensure that the results from both methods are comparable, parameters of the experimental and the simulation setup were chosen to be as similar as possible. From simulations, we saw that pyrene prefers a position inside the lipid membrane near the headgroups and has no tendency to diffuse from one monolayer of the membrane to the other. The results from simulation and NMR show that the normal of the molecular plane is aligned nearly perpendicular to the bilayer normal. The long axis of pyrene lies preferentially parallel to the bilayer normal within a range of +/-30 degrees . The results from the two different methods are remarkably consistent. The good agreement can be explained by the fact that the different kind of motions of a pyrene molecule are already averaged within a few nanoseconds, which is the timescale covered by the MD simulation.

FIGURE 1

The 10 deuterium atoms on the pyrene-d₁₀ molecule belong to three chemically different sites, and are denoted as 1, 2, and 3. In the MD simulation, all atoms are distinguishable, as indicated by primes in the figure. (A) The molecule-fixed xyz coordinate system is shown, with the z axis pointing into the plane, dash-dot-dash x axis for calculating S_xx, dash-dash y axis for calculating S_yy. (B) The quadrupolar coordinate system abc is illustrated for the deuteron at position 2. The a axis is along the C-D bond, and the c axis is parallel to the z axis, pointing into the plane.

FIGURE 2

Snapshot of the MD simulation starting configuration (left) at t = 0 ns, and the configuration (right) at t = 20 ns of the simulated box. The five pyrene molecules are shown as space filled in gray. Lipids are shown as sticks without hydrogen atoms, and with headgroup phosphates in black.

FIGURE 3

³¹P-NMR spectrum of an oriented sample of fully hydrated POPC containing 4% (mol/mol) pyrene-d₁₀.

FIGURE 4

²H-NMR spectra of an oriented sample of pyrene-d₁₀ in POPC. (Upper part) Bilayer normal oriented parallel to the static magnetic field direction. (Lower part) Bilayer normal oriented perpendicular to the magnetic field direction.

FIGURE 5

Positions of the center of mass of the five pyrene molecules in X (A), Y, (B) and Z directions (C) during the MD simulation. The lines in C indicate the headgroup region.

FIGURE 6

Area per lipid in nm² during the simulation period.

FIGURE 7

Orientation of the normal of the molecular plane with respect to the Z axis (S_zz) over the simulation time for all five pyrene molecules.

FIGURE 8

Normalized angle distribution of the angle between the molecular long axis 1/1′ and the Z axis (membrane normal). Values over the whole simulation time were taken into account.