Measurement of lateral diffusion rates in membranes by pulsed magnetic field gradient, magic angle spinning-proton nuclear magnetic resonance.

Membrane organization, including the presence of domains, can be characterized by measuring lateral diffusion rates of lipids and membrane-bound substances. Magic angle spinning (MAS) yields well-resolved proton nuclear magnetic resonance (NMR) of lipids in biomembranes. When combined with pulsed-field gradient NMR (rendering what is called "pulsed magnetic field gradients-MAS-NMR"), it permits precise diffusion measurements on the micrometer lengths scale for any substance with reasonably well-resolved proton MAS-NMR resonances, without the need of preparing oriented samples. Sample preparation procedures, the technical requirements for the NMR equipment, and spectrometer settings are described. Additionally, equations for analysis of diffusion data obtained from unoriented samples, and a method for correcting the data for liposome curvature are provided.

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

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.

Electrostatic contributions to indole-lipid interactions.

The role of electrostatic forces in indole-lipid interactions was studied by (1)H and (2)H NMR in ether- and ester-linked phospholipid bilayers with incorporated indole. Indole-ring-current-induced (1)H NMR chemical shifts of lipid resonances in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, and 1,2-di-O-octadecenyl-sn-glycero-3-phosphomethanol show a bimodal indole distribution, with indole residing at the upper hydrocarbon chain/glycerol region of the lipid and near the choline group, when present. (2)H NMR of indole-d(7)-incorporated lipid bilayers reveals that the former site is occupied by about two-thirds of the indole, which adopts a distinct preferred orientation with respect to the bilayer normal. The results suggest that the upper hydrocarbon chain/glycerol location is dictated by many factors, including interactions with the electric charges and dipoles, van der Waals interactions, entropic contributions, and hydrogen bonding. Indole diffusion rates are higher in lipids with ester bonds and lower in choline-containing lipids, suggesting that interactions between indole and carbonyl groups are of minor importance for lipid-indole association and that cation-pi interactions with choline drive the second indole location. Nuclear Overhauser effect spectroscopy cross-relaxation rates suggest a 30-ns lifetime for indole-lipid associations. These results may have important implications for sidedness and structural transitions in tryptophan-rich membrane proteins.

Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores.

Lipid bilayers were deposited inside the 0.2 microm pores of anodic aluminum oxide (AAO) filters by extrusion of multilamellar liposomes and their properties studied by 2H, 31P, and 1H solid-state NMR. Only the first bilayer adhered strongly to the inner surface of the pores. Additional layers were washed out easily by a flow of water as demonstrated by 1H magic angle spinning NMR experiments with addition of Pr3+ ions to shift accessible lipid headgroup resonances. A 13 mm diameter Anopore filter of 60 microm thickness oriented approximately 2.5 x 10(-7) mol of lipid as a single bilayer, corresponding to a total membrane area of about 500 cm2. The 2H NMR spectra of chain deuterated POPC are consistent with adsorption of wavy, tubular bilayers to the inner pore surface. By NMR diffusion experiments, we determined the average length of those lipid tubules to be approximately 0.4 microm. There is evidence for a thick water layer between lipid tubules and the pore surface. The ends of tubules are well sealed against the pore such that Pr3+ ions cannot penetrate into the water underneath the bilayers. We successfully trapped poly(ethylene glycol) (PEG) with a molecular weight of 8000 in this water layer. From the quantity of trapped PEG, we calculated an average water layer thickness of 3 nm. Lipid order parameters and motional properties are unperturbed by the solid support, in agreement with existence of a water layer. Such unperturbed, solid supported membranes are ideal for incorporation of membrane-spanning proteins with large intra- and extracellular domains. The experiments suggest the promise of such porous filters as membrane support in biosensors.

Multi-dimensional pulsed field gradient magic angle spinning NMR experiments on membranes.

The benefits of gradient techniques in the study of lipid membranes are demonstrated on a sample of 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC) liposomes embedded with ibuprofen. Most techniques from gradient NMR spectroscopy on solution samples are directly applicable to membrane samples subjected to magic angle spinning (MAS). Gradient-enhanced homo- and heteronuclear chemical shift correlation techniques were used to make resonance assignments. Gradient NOESY experiments provide insight into the location and dynamics of lipids, ibuprofen and water. Application of gradients not only reduces experiment time but also the t(1) noise in the multi-dimensional spectra. Diffusion measurements with pulsed field gradients characterize lateral movements of lipid and drug molecules in membranes. The theoretical framework for data analysis of MAS diffusion experiments on randomly oriented multilamellar liposomes is presented.

Lateral diffusion rates of lipid, water, and a hydrophobic drug in a multilamellar liposome.

The lateral diffusion constants of 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC), water, and ibuprofen were measured in multilamellar liposomes using pulsed field gradient magic-angle spinning (PFG-MAS) (1)H NMR. The analysis of diffusion data obtained in powder samples and a method for liposome curvature correction are presented. At 322 K POPC has a diffusion constant of (8.6 +/- 0.2) x 10(-12) m(2)/s when dehydrated (8.2 waters/lipid) and (1.9 +/- 0.1) x 10(-11) m(2)/s in excess water. The diffusion constant of water in dehydrated POPC was found to be (4.7 +/- 0.1) x 10(-10) m(2)/s. The radius of curvature is 21 +/- 2 microm for the dehydrated sample and 4.5 +/- 0.5 microm for POPC sample containing excess water. The activation energies of diffusion are 40.6 +/- 0.4 kJ/mole for dehydrated POPC, 30.7 +/- 0.9 kJ/mole for POPC with excess water, and 28.6 +/- 1.5 kJ/mole for water in dehydrated POPC. The diffusion constants and activation energies for a sample of POPC/ibuprofen/water (1:0.56:15) were also measured. The ibuprofen, which locates in the lipid-water interface, diffuses faster than POPC but has a slightly higher activation energy of lateral diffusion. Within certain restrictions, PFG-MAS NMR provides a useful method for characterizing membrane organization and mobility.