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.     

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.     

Solid state 13C NMR of unlabeled phosphatidylcholine bilayers: spectral assignments and measurement of carbon-phosphorus dipolar couplings and 13C chemical shift anisotropies.

The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.     

Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR.

Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.     

Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules

Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.     

Acidic phospholipid bicelles: a versatile model membrane system

With the aim of establishing acidic bicellar solutions as a useful membrane model system, we have used deuterium NMR spectroscopy to investigate the properties of dimyristoyl/dihexanoylphosphatidylcholine (DMPC/DHPC) bicelles containing 25% (w/w in H(2)O) of either dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG). The addition of the acidic lipid component to this lyotropic liquid crystalline system reduces its range of stability because of poor miscibility of the two dimyristoylated phospholipids. Compared to the neutral bicelles, which are stable at pH 4 to pH 7, acidic bicelles are stable only from pH 5.5 to pH 7. Solid-state deuterium NMR analysis of d(54)-DMPC showed similar ordering in neutral and acidic bicelles. Fully deuterated DMPS or DMPG is ordered in a way similar to that of DMPC. Study of the binding of the myristoylated N-terminal 14-residue peptide mu-GSSKSKPKDPSQRR from pp60(nu-src) to both neutral and acidic bicelles shows the utility of these novel membrane mimetics.     

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.     

Lateral diffusion of PEG-Lipid in magnetically aligned bicelles measured using stimulated echo pulsed field gradient 1H NMR

Lateral diffusion measurements of PEG-lipid incorporated into magnetically aligned bicelles are demonstrated using stimulated echo (STE) pulsed field gradient (PFG) proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Bicelles were composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) (q = DMPC/DHPC molar ratio = 4.5) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000) at 25 wt % lipid. 1H NMR STE spectra of perpendicular aligned bicelles contained only resonances assigned to residual HDO and to overlapping contributions from a DMPE-PEG 2000 ethoxy headgroup plus DHPC choline methyl protons. Decay of the latter's STE intensity in the STE PFG 1H NMR experiment (g(z) = 244 G cm(-1)) yielded a DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D = 1.35 x 10(-11) m2 s(-1). Hence, below the "mushroom-to-brush" transition, DMPE-PEG 2000 lateral diffusion is dictated by its DMPE hydrophobic anchor. D was independent of the diffusion time, indicating unrestricted lateral diffusion over root mean-square diffusion distances of microns, supporting the "perforated lamellae" model of bicelle structure under these conditions. Overall, the results demonstrate the feasibility of lateral diffusion measurements in magnetically aligned bicelles using the STE PFG NMR technique.     

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.     

Morphology of fast-tumbling bicelles: a small angle neutron scattering and NMR study

Bilayered micelles, or bicelles, which consist of a mixture of long- and short-chain phospholipids, are a popular model membrane system. Depending on composition, concentration, and temperature, bicelle mixtures may adopt an isotropic phase or form an aligned phase in magnetic fields. Well-resolved (1)H NMR spectra are observed in the isotropic or so-called fast-tumbling bicelle phase, over the range of temperatures investigated (10-40 degrees C), for molar ratios of long-chain lipid to short-chain lipid between 0.20 and 1.0. Small angle neutron scattering data of this phase are consistent with the model in which bicelles were proposed to be disk-shaped. The experimentally determined dimensions are roughly consistent with the predictions of R.R. Vold and R.S. Prosser (J. Magn. Reson. B 113 (1996)). Differential paramagnetic shifts of head group resonances of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC), induced by the addition of Eu(3+), are also consistent with the bicelle model in which DHPC is believed to be primarily sequestered to bicelle rims. Selective irradiation of the DHPC aliphatic methyl resonances results in no detectable magnetization transfer to the corresponding DMPC methyl resonances (and vice versa) in bicelles, which also suggests that DHPC and DMPC are largely sequestered in the bicelle. Finally, (1)H spectra of the antibacterial peptide indolicidin (ILPWKWPWWPWRR-NH(2)) are compared, in a DPC micellar phase and the above fast-tumbling bicellar phases for a variety of compositions. The spectra exhibit adequate resolution and improved dispersion of amide and aromatic resonances in certain bicelle mixtures.     

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.     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.     

Reinvestigation by phosphorus NMR of lipid distribution in bicelles

Mixtures of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC) in water form disks also called bicelles and different bilayer organizations when the mol ratio of the two lipids and the temperature are varied. The spontaneous alignment in a magnetic field of these bilayers above the transition temperature T(m) of DMPC is an attractive property that was successfully used to investigate protein structure by NMR. In this article, we have attempted to give an overview of all structural transformations of DMPC/DHPC mixtures that can be inferred from broad band (31)P-NMR spectroscopy between 5 and 60 degrees C. We show that above a critical temperature, T(v), perforated vesicles progressively replace alignable structures. The holes in these vesicles disappear above a new temperature threshold, T(h). The driving force for these temperature-dependent transformations that has been overlooked in previous studies is the increase of DHPC miscibility in the bilayer domain above T(m). Accordingly, we propose a new model (the "mixed bicelle" model) that emphasizes the consequence of the mixing. This investigation shows that the various structures of DMPC in the presence of increasing mol ratios of the short-chain DHPC is reminiscent of the observation put forward by several laboratories investigating solubilization and reconstitution of biological membranes.     

Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers: a FT-IR study

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers have been studied by Fourier-transform infrared spectroscopy (FT-IR) over the pressure range of 0.001-25 kbar. The results derived from the PRODAN C = O stretching band, the correlation field splitting of the methylene scissoring mode, and the methylene symmetric stretching mode as well as the absorption of the naphthalene ring show that in the sample of 4% (w/w) PRODAN in dimyristoyl-L-alpha-phosphatidylcholine (DMPC) at pH 6.8, most of the PRODAN molecules are embedded in the bilayers. In contrast, at pH 3.0, PRODAN was found to reside either on the membrane surface or dispersed in water. Compared to DMPC, egg yolk phosphatidylcholine (egg PC), which contains a substantial amount of unsaturated fatty acyl chains, is more susceptible to PRODAN permeation. The present study shows that the pressure dependence of the location of PRODAN in lipid membranes is different from that of tetracaine, a local anesthetic, in lipid bilayers. The model regarding the PRODAN location in lipid bilayers derived from the present infrared data has been compared with that obtained with previous fluorescence studies.     

Characterization of the structure and membrane interaction of the antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids

The mechanism of the association of human plasma apolipoprotein A-I (apo A-I) with the acidic phospholipids, dimyristoylphosphatidylglycerol (DMPG), egg yolk phosphatidylglycerol, and dioleoylphosphatidylserine as well as with the zwitterionic dimyristoylphosphatidylcholine (DMPC) has been studied using turbidimetry, circular dichroism, high-sensitivity differential scanning calorimetry, and electron microscopy. The association of apo A-I with multilamellar liposomes of acidic phospholipids is rapid over a broad temperature range at and above the temperature of the lipid gel to liquid crystalline transition, Tc. This is in contrast to zwitterionic phosphatidylcholine which recombines with apo A-I only over a narrow temperature range around Tc. The complex of apo A-I with DMPC denatures at elevated temperatures giving rise to a calorimetrically detectable transition. The temperature range and width of this transition is shown to be markedly dependent on the heating rate. This is again in contrast to apo A-I recombinants with DMPG which show no calorimetrically detectable thermal denaturation, at least in a temperature range up to 100 degrees C. Also circular dichroism data indicate high resistance of apo A-I to thermal unfolding in the presence of DMPG. It is concluded that the complexes of apo A-I with DMPC are thermodynamically stable only at temperatures near Tc, whereas above and below this temperature range the stability of these recombinants is determined by kinetic factors. In contrast, complexes of apo A-I with DMPG and other acidic phospholipids may be thermodynamically stable over a wide temperature range greater than or equal to Tc. In spite of these fundamental differences between zwitterionic and acidic phospholipids in their mode of association with apo A-I, the binding affinity and the morphology of the recombinants are similar. Both apo A-I X DMPC and apo A-I X DMPG complexes form lipoprotein particles having a discoidal shape.     

Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine > cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: The effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, S_mol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

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.