Structural Phase Behavior of High-Concentration,Alignable Biomimetic Bicelle Mixtures

The structures of bicelle mixtures composed of dimyristoyl and dihexanoyl phosphatidylcholines (DMPC and DHPC) with DMPC/DHPC molar ratios of 3.2 and 5 are characterized using polarized optical microscopy (POM) and small angle neutron scattering (SANS). Three phases, isotropic (I), chiral nematic (N^*) and smectic (S) are observed as temperature (T) varies from 10 to 70 °C. The structure of the magnetically alignable N^* phase, which was previously considered to be made up of discoidal micelles, is found to be composed of “ribbons”. Doping with the charged lipid, dimyristoyl phosphatidylglycerol (DMPG), which has the same 14:0 hydrocarbon chains as DMPC, results in a structural change of the aggregates where only the isotropic and smectic phases are observed. The smectic phase for the mixtures doped with DMPG is shear-alignable and follows one-dimensional swelling. However, at high-T zwitterionic DMPC/DHPC mixtures form multi-lamellar vesicles (MLV) with a relatively constant lamellar spacing of 66 Å, independent of water content.     

Photophysics of anthracene-indole systems in unilamellar vesicles of DMPC and POPC: Exciplex formation and temperature effects

The quenching of anthracene fluorescence by indole (IN), 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in dimiristoylphophatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. The studies were carried out at 25 degrees C in POPC vesicles and below (15 degrees C) and above (35 degrees C) the phase transition temperature (24 degrees C) of DMPC. A very efficient quenching of the anthracene fluorescence by IN and DMI in the lipid membrane is observed in all cases. It is less efficient in the case of Trp and IAA. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. Partition constants of the quenchers were obtained from the changes in the fluorescence emission of the indole moiety caused by the presence of the phospholipid. Using the partition constants bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. These corrected rate constants are lower than those in homogeneous solvents. In the case of DMPC values the gel phase are higher than in the liquid-crystalline phase. In the quenching by IN and DMI a new, red shifted, emission band appears which could be assigned to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp.     

Morphology of magnetically aligning DMPC/DHPC aggregates-perforated sheets, not disks

The morphology of DMPC/DHPC mixtures at total lipid concentration cL = 5% (w/w) and DMPC/DHPC ratio q approximately 3, doped with small amounts of DMPG or CTAB, was investigated. 31P NMR was used to identify the magnetically aligning phase, and cryo-transmission electron microscopy (cryo-TEM) was employed for structural characterization. Magnetic alignment was found to occur between approximately 30 and approximately 45 degrees C, and cryo-TEM showed that the magnetically aligning phase consisted of extended sheets with a lacelike structure. The aggregates are best described as intermediates between two-dimensional networks of flattened, highly branched, cylindrical micelles and lamellar sheets perforated by large irregular holes. DHPC most likely covers the edges of the holes, while DMPC makes up the bilayer bulk of the aggregates. However, 20-43% of the DHPC takes part in the bilayer, corresponding to 6-12% of the bilayer being made up of DHPC. This fraction increases with increasing temperature. At temperatures above 45 degrees C, the aligning phase collapses.     

Temperature driven annealing of perforations in bicellar model membranes

Bicellar model membranes composed of 1,2-dimyristoylphosphatidylcholine (DMPC) and 1,2-dihexanoylphosphatidylcholine (DHPC), with a DMPC/DHPC molar ratio of 5, and doped with the negatively charged lipid 1,2-dimyristoylphosphatidylglycerol (DMPG), at DMPG/DMPC molar ratios of 0.02 or 0.1, were examined using small angle neutron scattering (SANS), (31)P NMR, and (1)H pulsed field gradient (PFG) diffusion NMR with the goal of understanding temperature effects on the DHPC-dependent perforations in these self-assembled membrane mimetics. Over the temperature range studied via SANS (300-330 K), these bicellar lipid mixtures exhibited a well-ordered lamellar phase. The interlamellar spacing d increased with increasing temperature, in direct contrast to the decrease in d observed upon increasing temperature with otherwise identical lipid mixtures lacking DHPC. (31)P NMR measurements on magnetically aligned bicellar mixtures of identical composition indicated a progressive migration of DHPC from regions of high curvature into planar regions with increasing temperature, and in accord with the "mixed bicelle model" (Triba, M. N.; Warschawski, D. E.; Devaux, P. E. Biophys. J.2005, 88, 1887-1901). Parallel PFG diffusion NMR measurements of transbilayer water diffusion, where the observed diffusion is dependent on the fractional surface area of lamellar perforations, showed that transbilayer water diffusion decreased with increasing temperature. A model is proposed consistent with the SANS, (31)P NMR, and PFG diffusion NMR data, wherein increasing temperature drives the progressive migration of DHPC out of high-curvature regions, consequently decreasing the fractional volume of lamellar perforations, so that water occupying these perforations redistributes into the interlamellar volume, thereby increasing the interlamellar spacing.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

Magnetically aligned phospholipid bilayers with large q ratios stabilize magnetic alignment with high order in the gel and L(alpha) phases

The magnetic alignment behavior ofbicelles (magnetically alignable phospholipid bilayered membranes) as a function of the q ratio (1,2-dihexanoyl-sn-glycerol phosphatidylcholine/1,2-dimyristoyl-sn-glycerol phosphatidylcholine mole ratio) and temperature was studied by spin-labeled X-band electron paramagnetic resonance (EPR) spectroscopy and solid-state 2H and 31P NMR spectroscopy. Well-aligned bicelle samples were obtained at 45 degrees C for q ratios between 2.5 and 9.5 in both the EPR and NMR spectroscopic studies. The molecular order of the system, S(mol), increased as the q ratio increased and as the temperature decreased. For higher q ratios (> or = 5.5), bicelles maintained magnetic alignment when cooled below the main phase transition temperature (approximately 30 degrees C when in the presence of lanthanide cations), which is the first time, to our knowledge, that bicelles were magnetically aligned in the gel phase. For the 9.5 q ratio sample at 25 degrees C, S(mol) was calculated to be 0.83 (from 2H NMR spectra, utilizing the isotopic label perdeuterated 1,2-dimyristoyl-sn-glycerol phosphatidylcholine) and 0.911 (from EPR spectra utilizing the spin probe 3beta-doxyl-5alpha-cholestane). The molecular ordering of the high q ratio bicelles is comparable to literature values of S(mol) for both multilamellar vesicles and macroscopically aligned phospholipid bilayers on glass plates. The order parameter S(bicelle) revealed that the greatest degree of bicelle alignment was found at higher temperatures and larger q ratios (S(bicelle) = -0.92 for q ratio 8.5 at 50 degrees C).     

Membrane disordering induced by chloroform and carbon tetrachloride

We studied effects of chloroform and carbon tetrachloride on bilayer membranes of dimyristoyl-phosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (Egg-PC) by birefringence, dynamic light scattering and fluorescence methods. It is shown that interference light due to the membrane birefringence considerably decreases by addition of the organohalogen compounds for both lipid membranes, indicating a significant decrease in membrane order. In addition, results of dynamic light scattering and turbidity measurements show a rupture of multilamellar DMPC vesicles induced by addition of chloroform at concentrations above 0.2 v/v%. No rupture of the vesicles is observed within the limit of solubility of carbon tetrachloride in water, but excessive addition of carbon tetrachloride (above 0.2 v/v%) induces the vesicle rupture. Chain orientational order was estimated from the interference light intensity at low concentrations of the organohalogen compounds without the occurrence of the vesicle rupture. The estimation shows a monotonic decrease in the chain order with increasing the concentration. The decreases in DMPC chain order by chloroform and by carbon tetrachloride are about 17% at 0.2 v/v% and 23% at 0.05 v/v%, respectively. The reduction in the chain order is correlated with an increase in the membrane fluidity observed by excimer fluorescence of pyrene incorporated to the membrane. Behavior of membrane disordering of Egg-PC is approximately similar with that of DMPC. This implies the strong interaction between the organohalogen compounds and the lipid chains, whether or not the bilayer has the vacancy resulted from unsaturated double bonds and different chains in length. The results of this work suggest that damages of biological membranes by chloroform and tetrachloride are not only induced by a direct attack on proteins but also by a significant membrane disorder.     

Cholesterol modulates amiodarone-membrane interactions in model and native membranes

The effects of cholesterol, a lipid mostly found in the sarcolemmal membranes, on the interaction of amiodarone with synthetic models of dimyristoylphosphatidylcholine (DMPC) and with native models of mitochondria and brain microsomes was studied. Alterations on the structural order of lipids were assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probing the bilayer core, and of the propionic acid derivative 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA) probing the outer regions of the bilayer. As detected by the probes and according to classic observations, cholesterol progressively increased the molecular order in the fluid phase of DMPC. Additionally, it modulated the type and extension of amiodarone effects. For low cholesterol concentrations (≤10–15 mol%), amiodarone (50 μM) ordered DMPC bilayers and the effects were almost identical to those observed in pure DMPC. For higher cholesterol concentrations, amiodarone ordering effects decreased slightly and faded for cholesterol concentrations as high as 25 and 30 mol%, when detected by DPH-PA and DPH, respectively. Above these high cholesterol concentrations, a crossover from ordering to disordering effects of amiodarone was apparent, either in the upper region of the bilayer or the hydrophobic core. The effects of amiodarone in native membranes of mitochondria and brain microsomes, in which "native" cholesterol accounts for about 0 and 25 mol%, respectively, correlated reasonably with the results in models of synthetic lipids. There is a close relationship between cholesterol concentration and amiodarone effects, in either synthetic models or native model membranes. Therefore, it may be predicted that the lipid physicochemical properties regulated by cholesterol concentration will also modulate the effects of amiodarone in sarcolemma.     

大分子拥挤环境中脂肪酸影响磷脂囊泡相变的差示扫描量热研究

脂肪酸诱导的磷脂膜的热力学行为对于认识细胞内复杂的机制有着重要意义,而前人在研究脂肪酸与磷脂膜相互作用时大都在稀溶液中进行;拥挤环境下脂肪酸诱导磷脂膜的相变行为还未见报道。本文以二肉豆蔻酰磷脂酰胆碱(DMPC)构建囊泡模型,采用差示扫描量热法系统地研究了在不同浓度、不同分子量的聚乙二醇(PEG)拥挤环境中不同结构的脂肪酸对DMPC磷脂囊泡相变的影响。研究结果表明,在拥挤环境中,PEG对纯的磷脂囊泡相变的影响与大分子的分子量和浓度相关。对于脂肪酸/磷脂囊泡(FA/DMPC),PEG的存在对囊泡相变产生显著影响。在所考察的分子量和浓度范围内,PEG使FA/DMPC囊泡相变增加。短链饱和脂肪酸、不饱和脂肪酸原本使DPMC囊泡相变降低,但PEG缩小了降低幅度,甚至导致相变增加。进一步的研究表明,在大多数情况下,PEG对FA/DMPC的相变具有协作增强效应,且其影响均与大分子的分子量和浓度相关。另外,随着PEG浓度的升高,磷脂囊泡的协同单位数逐渐降低,表明拥挤环境会影响磷脂双分子层的均一性,使协同发生相变的分子数降低。本文的研究表明,大分子拥挤环境能够对扰动的磷脂双分子层起到一定的修复作用,这一现象在生物膜相关领域不可忽视。     

Urea Unfolding of Opsin in Phospholipid Bicelles†

Opsin is the unstable apo‐protein of the light‐activated G protein‐coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)/detergent bicelles, against urea‐induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far‐UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in α‐helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3‐[(cholamidopropyl)dimethylammonio]‐1‐propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin.     

Interaction of a poly(acrylic acid) oligomer with dimyristoylphosphatidylcholine bilayers

We studied the influence of 5 kDa poly(acrylic acid) (PAA) on the phase state, thermal properties, and lateral diffusion in bilayered systems of dimyristoylphosphatidylcholine (DMPC) using (31)P NMR spectroscopy, differential scanning calorimetry (DSC), (1)H NMR with a pulsed field gradient, and (1)H nuclear Overhauser enhancement spectroscopy (NOESY). The presence of PAA does not change the lamellar structure of the system. (1)H MAS NOESY cross-peaks observed for the interaction between lipid headgroups and polyion protons demonstrated only surface PAA-biomembrane interaction. Small concentrations of PAA (up to ～4 mol %) lead to the appearance of a new lateral phase with a higher main transition temperature, a lower cooperativity, and a lower enthalpy of transition. Higher concentrations lead to the disappearance of measurable thermal effects. The lateral diffusion coefficient of DMPC and the apparent activation energy of diffusion gradually decreased at PAA concentrations up to around 4 mol %. The observed effects were explained by the formation of at least two types of PAA-DMPC lateral complexes as has been described earlier (Fujiwara, M.; Grubbs, R. H.; Baldeschwieler, J. D. J. Colloid Interface Sci., 1997, 185, 210). The first one is characterized by a stoichiometry of around 28 lipids per polymer, which corresponds to the adsorption of the entire PAA molecule onto the membrane. Lipid molecules of the complex are exchanged with the "pure" lipid bilayer, with the lifetime of the complex being less than 0.1 s. The second type of DMPC-PAA complex is characterized by a stoichiometry of 6 to 7 lipids per polymer and contains PAA molecules that are only partially adsorbed onto the membrane. A decrease in the DMPC diffusion coefficient and activation energy for diffusion in the presence of PAA was explained by the formation of a new cooperative unit for diffusion, which contains the PAA molecule and several molecules of lipids.     

Unprecedented observation of days-long remnant orientation of phospholipid bicelles: a small-angle X-ray scattering and theoretical study

Nanometric bilayer-based self-assembled micelles commonly named as bicelles, formed with a mixture of long and short chains phosphatidylcholine lipids (PC), are known to orient spontaneously in a magnetic field. This field-induced orientational order strongly depends on the molecular structure of the phospholipids. Using small-angle X-ray scattering (SAXS), we performed detailed structural studies of bicelles and investigated the orientation/relaxation kinetics in three different systems: saturated-chain lipid bicelles made of DMPC (dimyristoyl PC)/DCPC (1,2-dicaproyl PC) with and without the added paramagnetic lanthanide ions Eu(3+), as well as bicelles of TBBPC (1-tetradecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-PC)/DCPC. The structural study confirmed the previous NMR studies, which showed that DMPC bicelles orient with the membrane normal perpendicular (defined here as "nematic" orientation) to the magnetic field, whereas they orient parallel (defined here as "smectic" orientation) to the magnetic field in the presence of Eu(3+). The TBBPC bicelles also show smectic orientation. Surprisingly, the orientational order induced in the magnetic field remains even after the magnetic field is removed, which allowed us to investigate the orientation and relaxation kinetics of different bicelle structures. We demonstrate that this kinetics is very different for all three types of bicelles at the same lipid concentration; DMPC bicelles (~40 nm diameter) with and without Eu(3+) orient faster than TBBPC bicelles (~80 nm diameter). However, for the relaxation, DMPC bicelles (nematic) lose their macroscopic orientation only after one hour, whereas both DMPC bicelles with Eu(3+) and TBBPC bicelles (smectic) remarkably stay oriented for up to several days! These results indicate that the orientation mechanism of these nanometric disks in the magnetic field is governed by their size, with smaller bicelles orienting faster than the larger bicelles. Their relaxation mechanism outside the magnetic field, however, is governed by the degree of ordering. Indeed, the angular distribution of oriented bicelles is much narrower for the bicelles with smectic orientation, and, consequently, they keep aligned for much longer time (days) than those with nematic ordering (hours) outside the magnetic field. The understanding of the orientation/relaxation kinetics, as well as the morphologies of these "molecular goniometers" at molecular and supramolecular levels, allows controlling such an unprecedented long-range and long-lived smectic ordering of nanodisks and opens a wide field of applications for structural biology or material sciences.     

Hydration dependent studies of highly aligned multilayer lipid membranes by neutron scattering

We investigated molecular motions on a picosecond timescale of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes as a function of hydration by using elastic and quasielastic neutron scattering. Two different hydrations corresponding to approximately nine and twelve water molecules per lipid were studied, the latter being the fully hydrated state. In our study, we focused on head group motions by using chain deuterated lipids. Information on in-plane and out-of-plane motions could be extracted by using solid supported DMPC multilayers. Our studies confirm and complete former investigations by Ko?nig et al. [J. Phys. II (France) 2, 1589 (1992)] and Rheinsta?dter et al. [Phys. Rev. Lett. 101, 248106 (2008)] who described the dynamics of lipid membranes, but did not explore the influence of hydration on the head group dynamics as presented here. From the elastic data, a clear shift of the main phase transition from the P(β) ripple phase to the L(α) liquid phase was observed. Decreasing water content moves the transition temperature to higher temperatures. The quasielastic data permit a closer investigation of the different types of head group motion of the two samples. Two different models are needed to fit the elastic incoherent structure factor and corresponding radii were calculated. The presented data show the strong influence hydration has on the head group mobility of DMPC.     

Discotic charge transfer twins Structure and mesophase behaviour of covalently linked triphenylenes and trinitrofluorenones

Discotic charge transfer twins, a novel class of discotic liquid-crystalline compounds were studied. These compounds consist of triphenylene units (as donors) which are chemically linked via flexible spacers of various lengths to trinitrofluorenone units (acting as acceptor). They display a liquid-crystalline phase over a wide temperature range extending up to 240-260°C. Based on X-ray analysis a structural model is proposed for the liquid-crystalline phase: the molecules are arranged in columns in such a way that mixed stacks occur, the intercolumnar packing possesses an orthorhombic symmetry. The neighbouring columns are connected along specific directions via flexible spacers which give rise to highly anisotropic structural properties of this columnar liquid-crystalline phase.     

Solid-state 2H NMR studies of the effects of cholesterol on the acyl chain dynamics of magnetically aligned phospholipid bilayers

We report the utilization of magnetically aligned phospholipid bilayers (bicelles) to study the effects of cholesterol in phospholipid bilayers for both chain perdeuterated DMPC and partially deuterated alpha-[2,2,3,4,4,6-d(6)]-cholesterol using (2)H solid-state NMR spectroscopy. The quadrupolar splittings at 40 degrees C were 25.5 and 37.7 kHz, respectively, for the 2,4-(2)H(eq) and 2,4-(2)H(ax) deuterons when the bilayer normal of the discs was aligned perpendicular to the static magnetic field. The quadrupolar splittings were doubled when Yb(3+) ions were added to flip the bicelles 90 degrees such that the bilayer normal was colinear with the magnetic field. The results suggest that cholesterol is incorporated into the bicelle discs. For chain perdeuterated DMPC-d(54), incorporated into DMPC-DHPC bicelle discs, the individual quadrupolar splittings of the methylene and methyl groups doubled on going from the perpendicular to the parallel alignment. Also, the presence of cholesterol increased the overall ordering of the acyl chains of the phospholipids. S(CD) (i) calculations were extracted directly from the (2)H quadrupolar splittings of the chain perdeuterated DMPC. The order parameter, S(CD) (i), calculations clearly indicated an overall degree of ordering of the acyl chains in the presence of cholesterol. We also noted a disordering effect at higher temperatures. This study demonstrates the ease with which (2)H order parameters can be calculated utilizing magnetically aligned phospholipid bilayers when compared with randomly dispersed membrane samples.     

Size and shape of fast-tumbling bicelles as determined by translational diffusion

In this study, the size and shape of an isotropic bicelle have been determined by measuring the translational diffusion as a function of the volume fraction of the lipids. A linear relation between the diffusion coefficients is obtained for both DMPC and DHPC in the bicelles. The slope of this linear function, which is strongly shape-dependent, is found to be different for the two molecules. This difference is direct evidence that the two molecules are not fully mixed in the bicelle. The shape- combined with the size-dependence of the diffusion coefficient allows us to calculate both the size and shape of the bicelle.     

Dynamic and 2D NMR studies on hydrogen-bonding aggregates of cholesterol in low-polarity organic solvents

Self-diffusion coefficients (D) are measured for normal (nondeuterated) and deuterated cholesterol-d(6) (C26 and C27 methyl groups deuterated) in 1-octanol, chloroform, and cyclohexane at concentrations of 1-700 mM by varying the impurity water concentration (>2 mM) and temperature (30-50 degrees C). The pulsed field gradient spin-echo (PGSE) (1)H and (2)H NMR were used, respectively, at 600 and 92 MHz. At 30 degrees C, the hydrodynamic radius (R) obtained at 20 mM from the D value and solvent viscosity is 5.09, 7.07, and 6.17 A, respectively, in 1-octanol, chloroform, and cyclohexane when the impurity water is negligible. The R value in 1-octanol is the smallest and comparable with the average length of the molecular axes for the cholesterol molecule. In 1-octanol, R is invariant against the concentration variation, whereas in chloroform, R is larger and increases almost linearly with cholesterol concentration. At the highest concentration, 700 mM, the R in chloroform is 13.5 and 16.7 A, respectively, when the impurity water is at negligible and saturated concentrations. The R value larger than that in hydrogen-bonding 1-octanol indicates that cholesterol forms an aggregate through hydrogen bonding. The aggregate structure is confirmed by comparing NOESY spectra in chloroform and 1-octanol. The NOESY analysis reveals the presence of one extra cross peak (C4-C19) in chloroform compared to 1-octanol. Because the carbon atoms related to the cross peak are close to the hydroxyl group (C3-OH), cholesterol molecules are considered to be not piled but are found to be OH-centered in the aggregate. This is supported also by larger rotational hydrodynamic radii measured on cholesterol deuterated at positions C2, C3, C4, and C6. This shows that the aggregate formation is driven by the hydrogen-bonding between cholesterol molecules.     

Bilayer in small bicelles revealed by lipid-protein interactions using NMR spectroscopy

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.     

Chain-melting phase transition and short-range molecular interactions in phospholipid foam bilayers

Occurrence of two-dimensional chain melting phase transition in foam bilayers was established for the first time. Microscopic horizontal foam bilayers [Newton black films (NBF)] were investigated by the microinterferometric method of Scheludko-Exerowa. The foam bilayers were formed from water-ethanol solutions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine (Egg PC) and samples of amniotic fluid (AF) at different temperatures. The influence of temperature on the foam bilayer thickness h(w) and on the critical concentration Cc for formation of foam bilayer was studied. It was shown that in the range of the main phase transition the temperature dependence of h(w) and C(c) changed specifically in the case of DMPC and DPPC foam bilayers. The thickness of the foam bilayers increased with decreasing temperature in the range of the main phase transition due to the melting of hydrocarbon tails of phospholipid molecules. These changes took place at the temperatures of the bulk chain-melting phase transitions, as determined by differential scanning calorimetry (DSC) for both aqueous, and water/ethanol DMPC, DPPC, and DPPC dispersions. An effect of the 'disperse medium' on h(w) was found for foam bilayers from DPPC. The results that foam bilayers could have different thickness at different temperatures disproved the current concept that NBF acquired constant thickness at concentrations higher than C(el,cr). The data for Cc were analysed on the basis of the hole-nucleation theory of bilayer stability of Kashchiev and Exerowa. This theory considered the amphiphile bilayer as a two-dimensional ordered system with short-range molecular interactions between the first neighbour molecules (as in a crystal). The short-range molecular interactions were presented by the parameter binding energy Q of an amphiphile molecule in the bilayer. The binding energy Q of two neighbouring phospholipids was calculated for the gel (30-60 kT) and liquid crystalline state (16-18 kT) of the bilayers from DMPC, DPPC, Egg PC, AF. Concentration/temperature phase diagram of DPPC foam bilayers that defined regions of gaseous (ruptured), gel and liquid crystalline foam bilayers were drawn. The values of Q obtained for various samples were very close and vary from 5.3 x 10(-20) to 9.4 x 10(-20) (approx. 13-22 kT) which indicated that in all cases the foam bilayers were in liquid-crystalline state. This is an important result since the parameter studied-threshold concentration (threshold dilution) is crucial for a very successful assessment of the risk for respiratory distress syndrome (RDS) in newborns and could be employed in medicine for assessment of other respiratory disturbances. It is to be expected that foam bilayers from phospholipids could be used as a model for investigation of short-range forces in biological structures, of interaction between membranes, etc.     

Magnetic resonance investigations of lipid motion in isotropic bicelles

The dynamics of DMPC in different isotropic bicelles have been investigated by NMR and EPR methods. The local dynamics were obtained by interpretation of 13C NMR relaxation measurements of DMPC in the bicelles, and these results were compared to EPR spectra of spin-labeled lipids. The overall size of the bicelles was investigated by PFG NMR translational diffusion measurements. The dynamics and relative sizes were compared among three different bicelles: [DMPC]/[DHPC] = 0.25, [DMPC]/[DHPC] = 0.5, and [DMPC]/[CHAPS] = 0.5. The local motion is found to depend much more strongly on the choice of the detergent, rather than the overall size of the bicelle. The results provide an explanation for differences in apparent dynamics for different peptides, which are bound to bicelles. This in turn determines under what conditions reasonable NMR spectra can be observed. A model is presented in which extensive local motion, in conjunction with the overall size, affects the spectral properties. An analytical expression for the size dependence of the bicelles, relating the radius of the bilayer region with physical properties of the detergent and the lipid, is also presented.