RESUMEN
Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200â¯nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Membrana Dobles de Lípidos/química , Proteínas de Transporte de Membrana/química , Microscopía por Crioelectrón , Epítopos/química , Escherichia coli/ultraestructura , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
The renin-angiotensin-aldosterone system (RAAS) plays a key role in the regulation of blood pressure. Renin is the rate limiting enzyme of the RAAS and aliskiren is a highly potent and selective inhibitor of the human renin. Renin is known to be active both in the circulating blood stream as well as locally, when bound to the (pro)-renin receptor ((P)RR). In this study we have investigated a possible mechanism of action of aliskiren, in which its accumulation in the plasma membrane is considered as an essential step for effective inhibition. Aliskiren's interactions with model membranes (cholesterol rich and poor) have been investigated by applying different complementary techniques: differential scanning calorimetry (DSC), Raman spectroscopy, magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy and small- and wide-angle X-ray scattering (SAXS and WAXS). In addition, in silico molecular dynamics (MD) calculations were applied for further confirmation of the experimental data. Aliskiren's thermal effects on the pre- and main transition of dipalmitoyl-phosphatidylcholine (DPPC) membranes as well as its topographical position in the bilayer show striking similarities to those of angiotensin II type 1 receptor (AT1R) antagonists. Moreover, at higher cholesterol concentrations aliskiren gets expelled from the membrane just as it has been recently demonstrated for the angiotensin receptor blocker (ARB) losartan. Thus, we propose that both the AT1R and the (P)RR-bound renin active sites can be efficiently blocked by membrane-bound ARBs and aliskiren when cholesterol rich membrane rafts/caveolae are formed in the vicinity of the receptors.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Amidas/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Membrana Celular/metabolismo , Fumaratos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Renina/metabolismo , Rastreo Diferencial de Calorimetría , Dominio Catalítico , Caveolas/metabolismo , Colesterol/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Renina/antagonistas & inhibidores , Dispersión del Ángulo Pequeño , Espectrometría Raman , Difracción de Rayos XRESUMEN
Losartan is an angiotensin II receptor antagonist mainly used for the regulation of high blood pressure. Since it was anticipated that losartan reaches the receptor site via membrane diffusion, the impact of losartan on model membranes has been investigated by small angle X-ray scattering. For this purpose 2-20 mol% losartan was incorporated into dimyristoyl-phosphatidylcholine (DMPC) and palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers and into their binary mixtures with cholesterol in the concentration range of 0 to 40 mol%. Effects of losartan on single component bilayers are alike. Partitioning of losartan into the membranes confers a negative charge to the lipid bilayers that causes the formation of unilamellar vesicles and a reduction of the bilayer thickness by 3-4%. Analysis of the structural data resulted in an estimate for the partial area of losartan, A(Los) ≈ 40 Å(2). In the presence of cholesterol, differences between the effects of losartan on POPC and DMPC are striking. Membrane condensation by cholesterol is retarded by losartan in POPC. This contrasts with DMPC, where an increase of the cholesterol content shifts the partitioning equilibrium of losartan towards the aqueous phase, such that losartan gets depleted from the bilayers from 20 mol% cholesterol onwards. This indicates (i) a chain-saturation dependent competition of losartan with lipid-cholesterol interactions, and (ii) the insolubility of losartan in the liquid ordered phase of PCs. Consequently, losartan's action is more likely to take place in fluid plasma membrane patches rather than in domains rich in cholesterol and saturated lipid species such as in membrane rafts.
Asunto(s)
Colesterol/química , Membrana Dobles de Lípidos/química , Losartán/química , Dimiristoilfosfatidilcolina/química , Membrana Dobles de Lípidos/metabolismo , Losartán/metabolismo , Microdominios de Membrana/metabolismo , Fosfatidilcolinas/químicaRESUMEN
We report on the characterization of mesostructured aerosol silica particles in the gas phase using in situ synchrotron small-angle X-ray scattering (SAXS) in order to unveil the influence of the basic production parameters. The investigated system was based on tetraethylorthosilicate (TEOS) as the inorganic precursor and on cetyltrimethyl-ammonium bromide (CTAB) as the surfactant. The heating temperature, surfactant to silicate ratio, and particle flow rate were thoroughly investigated, and for this purpose, an in-house-built aerosol reactor equipped with a special X-ray observation chamber was used. Complementary fine structural analysis was applied on dried deposits of the silica aerosols comprising direct Fourier transforms as well as simple two-phase model fits. This resulted in robust estimates for the silica wall thickness and surfactant core radius of the hexagonally ordered mesostructure. The particle shape and size distribution were examined by scanning electron microscopy (SEM). The quality of the inner nanostructure was revealed from an analysis of the peak width. The comparison of data from the gas phase and powder deposit shows that, in general, slower drying conditions (heating temperature about 80 °C) and a medium surfactant to Si ratio (about 0.14) lead to nanostructures of the best quality in terms of well-defined long-range organization.
Asunto(s)
Gases/química , Dióxido de Silicio/química , Difracción de Rayos X , Aerosoles , Electrones , Tamaño de la Partícula , Polvos , Dispersión del Ángulo Pequeño , TemperaturaRESUMEN
Valsartan is a marketed drug with high affinity to the type 1 angiotensin (AT1) receptor. It has been reported that AT1 antagonists may reach the receptor site by diffusion through the plasma membrane. For this reason we have applied a combination of differential scanning calorimetry (DSC), Raman spectroscopy and small and wide angle X-ray scattering (SAXS and WAXS) to investigate the interactions of valsartan with the model membrane of dipalmitoyl-phosphatidylcholine (DPPC). Hence, the thermal, dynamic and structural effects in bulk as well as local dynamic properties in the bilayers were studied with different valsartan concentrations ranging from 0 to 20 mol%. The DSC experimental results showed that valsartan causes a lowering and broadening of the phase transition. A splitting of the main transition is observed at high drug concentrations. In addition, valsartan causes an increase in enthalpy change of the main transition, which can be related to the induction of interdigitation of the lipid bilayers in the gel phase. Raman spectroscopy revealed distinct interactions between valsartan with the lipid interface localizing it in the polar head group region and in the upper part of the hydrophobic core. This localization of the drug molecule in the lipid bilayers supports the interdigitation view. SAXS measurements confirm a monotonous bilayer thinning in the fluid phase, associated with a steady increase of the root mean square fluctuation of the bilayers as the valsartan concentration is increased. At high drug concentrations these fluctuations are mainly governed by the electrostatic repulsion of neighboring membranes. Finally, valsartans' complex thermal and structural effects on DPPC bilayers are illustrated and discussed on a molecular level.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Tetrazoles/química , Valina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Algoritmos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Unión Competitiva , Rastreo Diferencial de Calorimetría , Cinética , Membrana Dobles de Lípidos/metabolismo , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Dispersión del Ángulo Pequeño , Espectrometría Raman , Temperatura , Tetrazoles/metabolismo , Termodinámica , Valina/química , Valina/metabolismo , Valsartán , Difracción de Rayos XRESUMEN
This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT(1)). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT(1) receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.
Asunto(s)
Imidazoles/metabolismo , Membrana Dobles de Lípidos/metabolismo , Pirrolidinas/metabolismo , Pirrolidinonas/química , Receptores de Angiotensina/metabolismo , 1,2-Dipalmitoilfosfatidilcolina , Rastreo Diferencial de Calorimetría , Imidazoles/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura Secundaria de Proteína , Protones , Pirrolidinas/química , Espectrometría Raman , Temperatura , Difracción de Rayos XRESUMEN
An in-house built aerosol generator setup for in situ gas phase studies of aerosol and nanoparticles is described. The aerosol generator with an ultrasonic ceramic disk mist maker provides high enough particle concentrations for structural gas phase analysis by synchrotron small angle x-ray scattering (for water approximately 4 x 10(8) droplets/s with a droplet size of approximately 2.5 microm). The working principle was proved by scattering of gold nanoparticles. For evaporation induced self-assembly studies of nanostructured particles, an additional thermal treatment chamber was included in the setup. The first on-line gas phase data with our setup for mesostructured silica particles are presented for different thermal treatments. Scanning electron microscope imaging revealed the average particle size to be approximately 1 microm. Furthermore, to quantify their internal nanostructure, diffraction experiments of deposited silica aerosols were carried out and the corresponding electron density map indicates a silica wall thickness of about 1 nm.
Asunto(s)
Aerosoles/química , Gases/química , Microfluídica/instrumentación , Nanopartículas/química , Nanopartículas/ultraestructura , Sonicación/instrumentación , Difracción de Rayos X/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Microfluídica/métodos , Transición de Fase , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodosRESUMEN
The effect of the surfactant content and hydration conditions in the phases of dioleoyl phosphatidylcoline (DOPC)/sodium dodecyl sulfate (SDS) mixtures was studied. To this end, surface X-ray diffraction experiments have been performed on bilayers of the mixtures deposited on hydrophobic silicon wafers by dip coating. To investigate the effect of relative humidity (RH) on bilayer organization, a humidity chamber with dry-wet air control was used, and RH values were fixed between 1 and 65%. Our results showed, in addition to the lamellar phase, a rhombohedral phase in mixtures at low hydration conditions (RH < 30%). The d spacing between lamellae increased with the RH and SDS content. This fact could be associated with a swelling effect that is probably due to the localization of water molecules between the polar headgroups of the DOPC and SDS forming the bilayers. The electron-density profiles calculated by Fourier reconstruction of the lamellar stacking for the different samples also confirmed this fact. In addition, the increase in d spacing could be related to the increase in the hydrophilic character of the mixture when the SDS content increases. The rhombohedral phase was more clearly observed in mixtures with high SDS content. Thus, the stalk structure of the rhombohedral phase could be facilitated because of the SDS contribution to inverse structures.
Asunto(s)
Membrana Dobles de Lípidos/química , Transición de Fase , Fosfatidilcolinas/química , Dodecil Sulfato de Sodio/química , Difracción de Rayos XRESUMEN
The collagen diffraction patterns of human aortas under uniaxial tensile test conditions have been investigated by synchrotron small-angle X-ray scattering. Using a recently designed tensile testing device the orientation and d-spacing of the collagen fibers in the adventitial layer have been measured in situ with the macroscopic force and sample stretching under physiological conditions. The results show a direct relation between the orientation and extension of the collagen fibers on the nanoscopic level and the macroscopic stress and strain. This is attributed first to a straightening, second to a reorientation of the collagen fibers, and third to an uptake of the increasing loads by the collagen fibers.
Asunto(s)
Aorta/fisiología , Colágeno/fisiología , Sincrotrones , Difracción de Rayos X/métodos , Aorta/química , Fenómenos Biomecánicos , Colágeno/química , Elasticidad , Humanos , Dispersión de Radiación , Resistencia a la Tracción , Difracción de Rayos X/instrumentaciónRESUMEN
We have determined the structural properties and bending fluctuations of fully hydrated phosphatidylcholine multibilayers in the fluid (Lalpha) phase, as well as the structure of the ripple (Pbeta') phase near the main phase transition temperature (TM) by x-ray diffraction. The number of carbons, nHC, per acyl chain of the studied disaturated lipids varied from 14 to 22. All lipids exhibit a nonlinear increase of the lamellar repeat distance d in the Lalpha phase upon approaching TM, known as "anomalous swelling." The nonlinear increase reduces with chain length, but levels off at a constant value of about 0.5 A for lipids with more than 18 hydrocarbons per chain. A detailed analysis shows that anomalous swelling has two components. One is due to an expansion of the water layer, which decreases with chain length and finally vanishes for nHC >18. The second component is due to a bilayer thickness increase, which remains unchanged in its temperature dependence, including a nonlinear component of about 0.5 A in the vicinity of TM. Thus, anomalous swelling above 18 hydrocarbons per chain is due to the pretransitional effects on the membrane only. These results are supported by a bending fluctuation analysis revealing increased undulations close to TM only for the short chain lipids. We have further calculated the electron density maps in the ripple phase and find no coupling of the magnitude of the ripple amplitude to the chain length effects observed in the Lalpha phase. Hence, in agreement with an earlier report by Mason et al. [Phys. Rev. E 63, 030902 (2001)] there is no connection between the formation of the ripple phase and anomalous swelling.
Asunto(s)
Biofisica/métodos , Fosfatidilcolinas/química , Carbono/química , Hidrocarburos/química , Membrana Dobles de Lípidos/química , Lípidos/química , Modelos Estadísticos , Temperatura , Agua , Difracción de Rayos XRESUMEN
We have observed a discontinuous unbinding transition of lipid bilayer stacks composed of phosphatidylethanolamine and phosphatidylglycerol using x-ray diffraction. The unbinding is reversible and coincides with the main (L(beta)-->L(alpha)) transition of the lipid mixture. Interbilayer interaction potentials deduced from the diffraction data reveal that the bilayers in the L(beta) phase are only weakly bound. The unbinding transition appears to be driven by an abrupt increase in steric repulsion resulting from increased thermal undulations of the bilayers upon entering the fluid L(alpha) phase.
Asunto(s)
Membrana Dobles de Lípidos/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Liposomas/química , Modelos Químicos , Difracción de Rayos XRESUMEN
1,2-diacyl-P-O-ethylphosphatidylcholines are synthetic cationic lipids that show some promising properties as nonviral DNA transfection agents. To gain further insight in the effects of the additional ethyl group in the headgroup region on the bilayer structure we systematically investigated a homologous series of fully hydrated ethylphosphatidylcholines with linear saturated chains (C14:0, C16:0, and C18:0) by small- and wide-angle X-ray diffraction. Our data show that all of them form multilamellar vesicles with chain interdigitated gel phases. Paying regard to the very importance of the liquid-crystalline phase in gene transfection, we applied the novel MCG method on high resolution X-ray diffraction data of the C16:0 derivative to be able to gain structural information on this phase. Comparison of this ethylphosphatidylcholine with its parent compound, the unesterified phosphatidylcholine, revealed that the major difference in the liquid-crystalline phase is the significantly reduced water layer between the bilayers for the cationic lipid. This may be one factor that contributes to the comparatively good DNA transfection efficiency of ethylphosphatidylcholines.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Difracción de Rayos X/métodosRESUMEN
We present a method for analyzing small angle x-ray scattering data on multilamellar phospholipid bilayer systems at full hydration. The method utilizes a modified Caillé theory structure factor in combination with a Gaussian model representation of the electron density profile such that it accounts also for the diffuse scattering between Bragg peaks. Thus the method can retrieve structural information even if only a few orders of diffraction are observed. We further introduce a procedure to derive fundamental parameters, such as area per lipid, membrane thickness, and number of water molecules per lipid, directly from the electron density profile without the need of additional volumetric measurements. The theoretical apparatus is applied to experimental data on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine liposome preparations.
Asunto(s)
Liposomas/química , Liposomas/metabolismo , Agua/metabolismo , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Dispersión de Radiación , Agua/química , Difracción de Rayos XRESUMEN
The magnetic resonance (MR) appearance of the weight-bearing ("loaded") and not-weight-bearing ("unloaded") regions in T(2)-weighted images of pig articular cartilage is different. On the hypothesis that this difference may be ascribed, at least in part, to a different collagen fibre organization in the two regions, this organization was studied using biochemical, histological, and X-ray diffraction methods. While the mean concentrations of collagen and of its cross-links were the same in the two regions, a regular small angle X-ray diffraction pattern was observed only for the habitually "loaded" tissue. It was also seen by light microscopy that the four typical functional zones were well displayed in the "loaded" cartilage whereas they were not clearly depicted in the "unloaded" tissue. Collagen presented a high concentration of fibrils forming an intricate and dense meshwork at the surface of both "loaded" and "unloaded" cartilage. A second zone of high collagen concentration was present at the upper layer of the deep zone of "loaded" cartilage. By contrast, this lamina of highly concentrated fibrils was lacking in "unloaded" cartilage and collagen fibrils appear thinner. Our study proves that the organization of collagen fibres is different for the "loaded" and "unloaded" regions of articular cartilage. It also suggests that this different organization may influence the MR appearance of the tissue. J. Exp. Zool. 287:346-352, 2000.
Asunto(s)
Cartílago Articular/ultraestructura , Colágeno/ultraestructura , Animales , Fenómenos Biomecánicos , Imagen por Resonancia Magnética , Porcinos/anatomía & histología , Soporte de PesoRESUMEN
Experimental evidence supporting the hypothesis of gel-liquid crystalline phase coexistence in the stable ripple phase of diacylphosphatidylcholines has been obtained from time-resolved X-ray small- (SAXS) and wide-angle diffraction (WAXS) in the millisecond to second time domain. The pretransition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) exhibits a thin lamellar liquid crystalline intermediate phase (designated Lalpha) if driven far away from equilibrium by an infrared temperature jump (T-jump) technique. The findings can be described by a two-step model. (1) Instantaneously with the T-jump, an anomalously thin lamellar liquid crystalline intermediate phase (d = 5.6-5.8 nm) forms, coexisting with the original gel-phase Lbeta'. Within the first seconds, the lamellar repeat distance of the intermediate increases to a value of about 6.7 nm. A closer examination of these kinetics reveals two relaxation components: a fast process, proceeding within tenths of a second, and a slow process, on the time scale of a few seconds. (2) Finally, both the liquid crystalline and the gel-phase relax into the stable ripple phase Pbeta'. The total process time of the transition is nearly independent of the addition of NaCl, but varies strongly with the chain length of the lecithin species.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Cristalización , Geles , Cinética , Modelos Moleculares , Conformación Molecular , Termodinámica , Difracción de Rayos XRESUMEN
By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lbeta phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12 degrees C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the 'Y-transition', is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1-->Lbeta transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 microl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lbeta phase at 50 degrees C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lbeta phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes.
Asunto(s)
Membrana Dobles de Lípidos/química , Fosfatidiletanolaminas/química , Temperatura , Rastreo Diferencial de Calorimetría , Frío , Cristalización , Densitometría/métodos , Geles , Termodinámica , Difracción de Rayos X/métodosRESUMEN
The effects of alkali chlorides on phosphatidylcholine-water bilayer systems in the Lalpha-phase were investigated by using small- and wide-angle X-ray scattering. The ternary system LiCl-POPC-H2O under isothermal conditions has shown that above Li+/POPC molar ratios of 0.1 and a lipid concentration above 5% (w/w), a splitting of the lamellar Bragg diffraction peaks into discrete components indicates a phase separation into different lamellar liquid crystalline (smectic A) phases. It is also shown that in saturated distearoyl phosphatidylcholine and in egg phosphatidylcholine, alkali chlorides induce Lalpha-phase separation. The number and repeat distance of the coexisting lamellar phases depend on the nature and concentration of the alkali chloride, the concentration of the phosphatidylcholine, and the degree of the acyl chain unsaturation.
Asunto(s)
Cloruros/química , Fosfatidilcolinas/química , Agua/química , Fenómenos Químicos , Química Física , Cristalización , Concentración de Iones de Hidrógeno , Cloruro de Litio/química , Dispersión de Radiación , Rayos XRESUMEN
The double-focusing high-flux wiggler beamline dedicated to small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) at ELETTRA has gone into user operation recently. It has been designed specifically for time-resolved studies of non-crystalline and fibrous materials in the submillisecond time scale, and has been optimized for small-angle scattering measurements. An overview of the beamline status and of some representative results, highlighting the performance of the SAXS beamline, are given.
RESUMEN
Time-resolved small-angle X-ray diffraction of liquid-crystalline phospholipid-water systems under temperature or pressure jump conditions has demonstrated the existence of an ordered, intermediate L alpha phase, with a sub-second lifetime, designated as the L* alpha-phase. The lamellar repeat spacing is, universally, 0.3 nm smaller than that of the parent phase, irrespective of the lipid composition and of the jump conditions, provided that the jump leads to a net volume expansion of the phase. The presence of salts, most notably LiCl, leads to a prolongation of the lifetime. The results suggest a non-monotonic potential function for the interbilayer water thickness.
Asunto(s)
Membranas Artificiales , Fosfolípidos/química , Agua/química , Difracción de Rayos XRESUMEN
A new lamellar gel phase (L beta *) with expanded lamellar period was found at low temperatures in dihexadecylphosphatidylethanolamine (DHPE) and dipalmitoylphosphatidylethanolamine (DPPE) dispersions in concentrated sucrose solutions (1-2.4 M). It forms via a cooperative, relatively broad transition upon cooling of the L beta gel phase of these lipids. According to the X-ray data, the transformation between L beta and L beta * is reversible, with a temperature hysteresis of 6-10 degrees C and a transition width of about 10 degrees C. No specific volume changes and a very small heat absorption of about 0.05 kcal/mol accompany this transition. The L beta *-L beta transition temperature strongly depends on the disaccharide concentration. From a value of about 10 degrees C below the melting transition of DHPE, it drops by 25 degrees C with decrease of sucrose concentration from 2.4 M to 1 M. The low-temperature gel phase L beta * has a repeat spacing by 8-10 A larger than that of the L beta gel phase and a single symmetric 4.2 A wide-angle peak. It has been observed in 1, 1.25, 1.5 and 2.4 M solutions of sucrose, but not in 0.5 M of sucrose. The data clearly indicate that the expanded lamellar period of the L beta * phase results from a cooperative, reversible with the temperature, increase of the interlamellar space of the L beta gel phase. Other sugars (trehalose, maltose, fructose, glucose) induce similar expanded low-temperature gel phases in DHPE and DPPE. The L beta * phase is osmotically insensitive. Its lamellar period does not depend on the sucrose concentration, while the lattice spacings of the L alpha, L beta and HII phases decrease linearly with increase of sucrose concentration. Another notable sugar effect is the induction of a cubic phase in these lipids. It forms during the reverse HII-L alpha transition and coexists with the L alpha phase in the whole temperature range between the HII and L beta phases. The cubic phase has only been observed at sucrose concentrations of I M and above. In accordance with previous data, sucrose suppresses the L alpha phase in both lipids and brings about a direct L beta-HII phase transition in DHPE. A raid, reversible gel-subgel transformation takes place at 17 degrees C in both DPPE and DHPE. Its properties do not depend on the sucrose concentration. The observed new effects of disaccharides on the properties of lipid dispersions might be relevant to their action as natural protectants.