Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS)

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by ³¹P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7°, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0° toward 54.7°. Thus the relative sign of dipolar and scalar couplings can be determined.     

Delta-opiate DPDPE in magnetically oriented phospholipid micelles: binding and arrangement of aromatic pharmacophores

D-Penicillamine(2,5)-enkephalin (DPDPE) is a potent opioid peptide that exhibits a high selectivity for the delta-opiate receptors. This zwitterionic peptide has been shown, by pulsed-field gradient 1H NMR diffusion studies, to have significant affinity for a zwitterionic phospholipid bilayer. The bilayer lipid is in the form of micelles composed of dihexanoylphosphatidylcholine (DHPC) and dimyristoylphosphatidylcholine (DMPC) mixtures, where the DMPC forms the bilayer structure. At high lipid concentration (25% w/w) these micelles orient in the magnetic field of an NMR spectrometer. The resulting 1H-13C dipolar couplings and chemical shift changes in the natural abundance 13C resonances for the Tyr and Phe aromatic rings were used to characterize the orientations in the bilayer micelles of these two key pharmacophores.     

Opsin stability and folding: modulation by phospholipid bicelles

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-α-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to  70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.     

Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO.

Buffered mixtures of the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) and dimyristoylphosphatidylcholine (DMPC) orient in the presence of a strong magnetic field over a wide range of water contents (at least 65-85%) and CHAPSO:DMPC molar ratios (typically 1:10-1:3). 31P NMR studies show that the phospholipid in such mixtures is oriented with its director axis perpendicular to the magnetic field. 31P and 2H NMR results also suggest that the structure and dynamics of the DMPC molecules are similar to that of pure phospholipids existing in the liquid crystalline (L alpha) bilayer phase. The ability of 1:5 CHAPSO:DMPC samples to orient is highly tolerant of large changes in temperature, pH, and ionic strength, as well as to the addition of substantial amounts of charged amphiphiles or soluble protein. However, 2H NMR studies of deuterated beta-dodecyl melibiose (DD-MB) solubilized in the system indicate the head group conformation and/or dynamics of this glycolipid analogue is dependent upon the CHAPSO concentration. Despite the latter results, the orientational versatility of the system, together with the nondenaturing properties of CHAPSO, makes this system useful in spectroscopic studies of membrane-associated phenomena.     

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of ¹H, ¹³C or ¹⁵N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 °C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.     

Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values

It is demonstrated that mixtures of ditetradecyl- phosphatidylcholine or didodecyl-phoshatidylcholine and dihexyl- phosphatidylcholine in water form lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl- phosphatidylcholine (DHPC). The carboxy-ester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid- or base-catalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3–10.4pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range.     

Orientational behavior of phosphatidylcholine bilayers in the presence of aromatic amphiphiles and a magnetic field.

A number of aromatic-containing additives which can influence the orientation of fragments of lipid bilayer membranes by a magnetic field have been investigated. Two properties of these additives prove important: (1) sufficient detergency to facilitate reorganization of bilayer components and (2), sufficient anisotropy in magnetic susceptibility the preferred direction of fragment orientation. Triton X-100 is identified as effective in terms of facilitating magnetic field ordering of bilayer fragments but does not alter the preferred direction of orientation. A combination of the detergent CHAPSO (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate) and the aromatic alcohol 1-naphthol facilitates both ordering and alters the preferred direction of bilayer orientation. As mixtures of dimyristoylphosphatidylcholine (DMPC) and CHAPSO, which orient with bilayer normals perpendicular to the magnetic field, were titrated with 1-naphthol, the assemblies underwent transitions, first to random orientation, and then to an orientation with bilayer normals parallel to the field. Based on temperature-induced phase transitions and the extent of motional averaging of the 31P shielding tensor of the DMPC headgroup, the DMPC in these oriented samples appears to maintain a bilayer morphology during transitions. The insight provided in this study regarding factors which influence fragment stability and orientation lays the groundwork for the design of improved field-oriented media for spectroscopic investigation of membrane components.     

Study of phospholipid structure by 1H, 13C, and 31P dipolar couplings from two-dimensional NMR.

Various motionally averaged 31P-1H, 13C-1H, 1H-1H, and 31P-13C dipolar couplings were measured for natural-abundance and unoriented phosphocholine in the L alpha phase. The couplings were obtained and assigned by a variety of advanced and partly novel two-dimensional solid-state NMR experiments. Whereas 31P-1H and 31P-13C dipolar couplings provide long-range structural constraints, geminal 1H-1H couplings and the signs of 13C-1H couplings are important new elements in a segmental order-tensor analysis of the lipid headgroup and glycerol backbone. The implications of these measured dipolar couplings for the conformational exchange of the lipid headgroup and the bending of the headgroup from the glycerol backbone are discussed. These dipolar couplings are also analyzed semiquantitatively in terms of the segmental order tensor.     

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.     

Phospholipid vesicle formation using nonionic detergents with low monomer solubility. Kinetic factors determine vesicle size and permeability

The method developed previously for formation of unilamellar vesicles from mixed micelles of egg lecithin and octyl glucoside [Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., & Reynolds, J. A. (1981) Biochemistry 20, 833-840] has been extended to allow for (1) use of nonionic detergents with much lower critical micelle concentrations and (2) variation in the time course of detergent removal. The results demonstrate the importance of kinetic factors, especially in the determination of vesicle size: initially formed vesicles are small, but the size increases slowly thereafter if detergent is not removed too quickly. Vesicle size remains fixed when the molar detergent/lipid ratio falls below about 1/1, and detergent removal becomes increasingly difficult thereafter, presumably because flip-flop of detergent from the inner to the outer leaflet of the bilayer membrane is very slow. Residual detergent (to about 25 mol %) has surprisingly little effect on anion permeability but increases cation permeability to the point where the normal discrimination between anions and cations (in pure lipid vesicles) is lost. Detergent added to initially detergent-free vesicles readily partitions into vesicular membranes (presumably only into the outer leaflet) and has a qualitatively similar effect on permeability. Vesicles produced by this method, regardless of residual detergent level, were found to be predominantly unilamellar: no multilamellar liposomes or other lipid aggregates could be detected within the accuracy of the methods employed.     

High resolution 13C NMR spectra on oriented lipid bilayers: from quantifying the various sources of line broadening to performing 2D experiments with 0.2-0.3 ppm resolution in the carbon dimension

¹³C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1–2 ppm, although T₂ measurements indicate that 0.1–0.2 ppm could be obtained. We have prepared a DMPC – ¹³C₄-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90° (or of the magic angle) with B₀. We have measured T₂s, CSAs, and linewidths for the choline ¹³C--methyl, the cholesterol-C₄ carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of B₀ field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and ¹³C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of ±0.30° ), ¹³C linewidth of 0.2–0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90°, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 ± 2 Hz between the choline methyl carbons was determined.     

Raman spectroscopic studies of the packing properties of mixed dihexadecyl- and dipalmitoylphosphatidylcholine bilayer dispersions

X-ray diffraction studies suggest the existence of two separate gel phases for mixed dihexadecylphosphatidylcholine (DHPC)/dipalmitoylphosphatidylcholine (DPPC) bilayers [Kim, J. T., Mattai, J., & Shipley, G. G. (1987) Biochemistry 26, 6599-6603; Lohner, K., Schuster, A., Degovics, G., Müller, K., & Laggner, P. (1987) Chem. Phys. Lipids 44, 61-70]. In one gel phase the lipid chains are interdigitated, while the other gel phase exhibits the conventional bilayer form. We use Raman spectroscopy to provide a detailed molecular analysis of the intermolecular and intramolecular interactions of the DHPC and DPPC molecules within these mixed bilayers. Observation of the methylene chain C-H stretching modes of DHPC and the methylene chain C-D stretching modes of DPPC-d62 for various mixed DHPC/DPPC-d62 bilayers enables the packing characteristics and conformational order of each lipid to be monitored separately. The spectral data indicate that the packing properties of DPPC-d62 in the mixed-lipid bilayers remain relatively unchanged, while the intramolecular and intermolecular properties of DHPC change dramatically as a function of the composition of the DHPC/DPPC-d62 mixed bilayer. This is consistent with a model based upon the existence of three characteristic lipid types for the mixed-lipid system, namely, domains of pure DPPC-d62 and pure DHPC species with interface lipids or boundary regions between the bulk domains.     

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.     

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

Phospholipid containing mixed micelles. Characterization of diheptanoyl phosphatidylcholine (DHPC) and sodium dodecyl sulfate and DHPC and dodecyl trimethylammonium bromide

Mixed micelles of l,2-diheptanoyl-sn-grycero-3-phosphocholine (DHPC) with ionic detergents were prepared to develop well characterized substrates for the study of lipolytic enzymes. The aggregates that formed on mixing DHPC with the anionic surfactant sodium dodecyl sulfate (SDS) and with the positively charged dodecyl trimethylammonium bromide (DTAB) were investigated using time-resolved fluorescence quenching (TRFQ) to determine the aggregation numbers and bimolecular collision rates, and electron spin resonance (ESR) to measure the hydration index and microviscosity of the micelles at the micelle-water interface. Mixed micelles between the phospholipid and each of the detergents formed in all compositions, yielding interfaces with varying charge, hydration, and microviscosity. Both series of micelles were found to be globular up to 0.7 mole fraction of DHPC, while the aggregation numbers varied within the same concentration range of the components less than 15%. Addition of the zwitterionic phospholipid component increased the degree of counterion dissociation as measured by the quenching of the fluorescence of pyrene by the bromide ions bound to DHPC/DTAB micelles, showing that at 0.6 mole fraction of DHPC 80% of the bromide ions are dissociated from the micelles. The interface water concentration decreased significantly on addition of DHPC to each detergent. For combined phospholipid and detergent concentration of 50 mM the interface water concentration decreased, as measured by ESR of the spin-probes, from 38.5 M/L of interface volume in SDS alone to 9 M/L when the phospholipid was present at 0.7 mole fraction. Similar addition of DHPC to DTAB decreased the interfacial water concentration from 27 M/L to 11 M/L. Determination of the physicochemical parameters of the phospholipid containing mixed micelles here presented are likely to provide important insight into the design of assay systems for kinetic studies of phospholipid metabolizing enzymes.     

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.     

Conformational studies of human milk oligosaccharides using (1)H-(13)C one-bond NMR residual dipolar couplings

1H-(13)C one-bond dipolar coupling values were measured for natural abundance samples of the human milk oligosaccharides "lacto-N-fucopentaose" (LNF-1 LNF-2, and LNF-3), "lacto-N-difucohexaose" (LND-1), "lacto-N-tetraose" (LNT), and "lacto-N-neo-tetraose" (LNnT), four of which have Lewis blood group epitopes. Each oligosaccharide was dissolved in a 7.5% solution of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelle liquid crystals oriented in the NMR magnetic field. The dipolar coupling data and NOE were fitted to conformational models with calculations of an optimum orientation tensor which best represents the dipolar coupling values for a fragment hypothesized to adopt a single conformation. In the case of LNF-1, LNF-2, LNF-3, and LND-1, the models confirm previous conformational models for the Lewis epitopes based on NOE and molecular dynamics simulations. Extensions of the model provided new structural data for the remaining residues. In all cases, upper limits for the errors in the glycosidic angles of the models were estimated. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.     

Atomistic Simulations of Bicelle Mixtures

Mixtures of long- and short-tail phosphatidylcholine lipids are known to self-assemble into a variety of aggregates combining flat bilayerlike and curved micellelike features, commonly called bicelles. Atomistic simulations of bilayer ribbons and perforated bilayers containing dimyristoylphosphatidylcholine (DMPC, di-C₁₄ tails) and dihexanoylphosphatidylcholine (DHPC, di-C₆ tails) have been carried out to investigate the partitioning of these components between flat and curved microenvironments and the stabilization of the bilayer edge by DHPC. To approach equilibrium partitioning of lipids on an achievable simulation timescale, configuration-bias Monte Carlo mutation moves were used to allow individual lipids to change tail length within a semigrand-canonical ensemble. Since acceptance probabilities for direct transitions between DMPC and DHPC were negligible, a third component with intermediate tail length (didecanoylphosphatidylcholine, di-C₁₀ tails) was included at a low concentration to serve as an intermediate for transitions between DMPC and DHPC. Strong enrichment of DHPC is seen at ribbon and pore edges, with an excess linear density of ∼3 nm⁻¹. The simulation model yields estimates for the onset of edge stability with increasing bilayer DHPC content between 5% and 15% DHPC at 300 K and between 7% and 17% DHPC at 323 K, higher than experimental estimates. Local structure and composition at points of close contact between pores suggest a possible mechanism for effective attractions between pores, providing a rationalization for the tendency of bicelle mixtures to aggregate into perforated vesicles and perforated sheets.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

31P NMR first spectral moment study of the partial magnetic orientation of phospholipid membranes.

Structural data can be obtained on proteins inserted in magnetically oriented phospholipid membranes such as bicelles, which are most often made of a mixture of long and short chain phosphatidylcholine. Possible shapes for these magnetically oriented membranes have been postulated in the literature, such as discoidal structures with a thickness of one bilayer and with the short acyl chain phosphatidylcholine on the edges. In the present paper, a geometrical study of these oriented structures is done to determine the validity of this model. The method used is based on the determination of the first spectral moment of solid-state (31)P nuclear magnetic resonance spectra. From this first moment, an order parameter is defined that allows a quantitative analysis of partially oriented spectra. The validity of this method is demonstrated in the present study for oriented samples made of DMPC, DMPC:DHPC, DMPC:DHPC:gramicidin A and adriamycin:cardiolipin.