Hydration of POPC bilayers studied by 1H-PFG-MAS-NOESY and neutron diffraction

Abstract

The stability of lipid bilayers is ultimately linked to the hydrophobic effect and the properties of water of hydration. Magic angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) with application of pulsed magnetic field gradients (PFG) was used to study the interaction of water with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers in the fluid phase. NOESY cross-relaxation between water and polar groups of lipids, but also with methylene resonances of hydrophobic hydrocarbon chains, has been observed previously. This observation led to speculations that substantial amounts of water may reside in the hydrophobic core of bilayers. Here, the results of a quantitative analysis of cross-relaxation in a lipid 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC)/water mixture are reported. Coherences were selected via application of pulsed magnetic field gradients. This technique shortens acquisition times of NOESY spectra to 20 min and reduces t (1)-spectral noise, enabling detection of weak crosspeaks, like those between water and lipids, with higher precision than with non-gradient NOESY methods. The analysis showed that water molecules interact almost exclusively with sites of the lipid-water interface, including choline, phosphate, glycerol, and carbonyl groups. The lifetime of lipid-water associations is rather short, on the order of 100 ps, at least one order of magnitude shorter than the lifetime of lipid-lipid associations. The distribution of water molecules over the lipid bilayer was measured at identical water content by neutron diffraction. Water molecules penetrate deep into the interfacial region of bilayers but water concentration in the hydrophobic core is below the detection limit of one water molecule per lipid, in excellent agreement with the cross-relaxation data.

Fig. 1

Contour plot of a two-dimensional NOESY spectrum of the POPC/water dispersion, recorded at a mixing time of 300 ms and a temperature of 10°C. Contour levels with positive intensity are shown in earthtone colors and negative intensities in blue. Resonance assignments are provided in Fig. 2.

Fig. 2

Rows of two-dimensional NOESY spectra, recorded as a function of mixing time, showing water-to-lipid crosspeaks. The top spectrum is the 1D projection of a NOESY experiment, recorded at a mixing time of 5 ms. It shows the diagonal resonances, attenuated 256-fold. Resonance assignments are provided as numbers. The region of the intense water resonance was blended out for clarity.

Fig. 3

A) Normalized, per-proton cross-relaxation rates of magnetization transfer from water-to-lipid protons, Γ_water-lipid. Resonance assignments are provided as numbers (see Fig.2). Cross-relaxation rates to protons of choline- and glycerol groups are low and positive. All cross-relaxation rates to hydrocarbon chain resonances are very low. B) Normalized, per-proton cross-relaxation rates of magnetization transfer from the choline methyl- to other lipid protons, Γ_{N(CH3)3-lipid}. Generally, those rates are negative. Please note that the y-scales of graphs A and B differ by one order of magnitude. Cross-relaxation rates from choline γ to resonances of the lipid-water interface are high. Weaker rates to lipid hydrocarbon chain resonances are observed as well. They reveal the tumultuous disorder in fluid lipid bilayers.

Fig. 4

Scattering length density (SLD) distributions of POPC and water along the bilayer normal, measured at a relative humidity of 93% and ambient temperature. The value z = 0 Å corresponds to the bilayer center. The water density (blue) was calculated from the difference of density profiles recorded as a function of ²H₂O content in the water of hydration (see Results section for details).

Fig. 5

Mean water distributions, determined as the difference scattering length densities of POPC measured in ²H₂O and H2O, respectively. The colors from dark to light blue correspond to hydration of POPC bilayers at relative the relative humidities 66%, 76%, 86%, and 93%. With increasing water content, density of water and the thickness of the water layer increase. The center of the water layer moves somewhat closer to the bilayer center with increasing hydration. The latter is a reflection of a thinning of bilayers with increasing water content.

Fig. 6

Dependence of NOESY cross-relaxation rates on the correlation time, τ, of reorientation of the vector connecting the interacting protons. The dependence was calculated for a proton resonance frequency of 500 MHz. At correlation times τ<400 ps, cross-relaxation rates are positive. They reach a local maximum at τ≈100 ps. At correlation times τ>400 ps, cross-relaxation rates are negative and increase steadily with increasing length of correlation times (see Discussion section for details).