Determination of fluid--solid transitions in model protein solutions using the histogram reweighting method and expanded ensemble simulations

Protein crystallization conditions are usually identified by empirical screening methods because of the complexity of the process, such as the existence of nonequilibrium phases and the different crystal forms that may result from changes in solution conditions. Here the crystallization of a model protein is studied using computer simulation. The model consists of spheres that have both an isotropic interaction of short range and anisotropic interactions between patch-antipatch pairs. The free energy of a protein crystal is calculated using expanded ensemble simulations of the Einstein crystal, and NpT-Monte Carlo simulations with histogram reweighting are used to determine the fluid-solid coexistence. The histogram reweighting method is also used to trace out the complete coexistence curve, including multiple crystal phases, with varying reduced temperature, which corresponds to changing solution conditions. At a patch-antipatch interaction strength five times that of the isotropic interaction, the protein molecules form a stable simple cubic structure near room temperature, whereas an orientationally disordered face-centered-cubic structure is favored at higher temperatures. The anisotropic attractions also lead to a weak first-order transition between orientationally disordered and ordered face-centered-cubic structures at low temperature, although this transition is metastable. A complete phase diagram, including a fluid phase, three solid phases, and two triple points, is found for the six-patch protein model. A 12-patch protein model, consistent with the face-centered-cubic structure, leads to greater thermodynamic stability of the ordered phase. Metastable liquid-liquid phase equilibria for isotropic models with varying attraction tails are also predicted from Gibbs ensemble simulations.     

Films of novel mesogenic molecules at air-water and air-solid interfaces

Discotic molecules are known to form highly anisotropic structures at the air-water (A-W) interface. We have studied two novel ionic discotic mesogenic molecules, viz., pyridinium tethered with hexaalkoxytriphenylene with bromide counterion (Py-Tp) and imidazolium tethered with hexaalkoxytriphenylene with bromide counterion (Im-Tp) at A-W and air-solid interfaces. The monolayer phases were investigated at the A-W interface employing surface manometry and Brewster angle microscopy techniques. They indicate a uniform monolayer phase which shows negligible hysteresis on expanding and compressing. Also, in both the systems the collapsed state completely reverts to the monolayer state. These monolayer films transferred at different surface pressures by Langmuir-Blodgett technique were studied by employing atomic force microscopy. The topographies of these films transferred at the low and high surface pressure region of the isotherm indicate a transformation of the monolayer from face-on to edge-on structure.     

Bilayer lipid membrane formation on a chemically modified S-layer lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ～6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).     

Interactions of lipases with lipid monolayers. Facts and questions

Among the proteins, lipolytic enzymes provide a valuable model for studying protein-lipid interactions. Lipases having a catalytic action which is strictly dependent upon the presence of a lipid interface were used in the present study in order to gain better insight into protein-lipid interactions. Most of the data presented here were obtained using the monolayer technique, by recording (either independently or simultaneously) the lipolytic activity, the amount of protein adsorbed to the lipid monolayer, and the surface pressure variations following protein adsorption. Several non-enzymatic proteins were used as controls in order to determine how lipase behaviour differs from that of other proteins. At all initial surface pressures tested, with zwitterionic monolayers, a good correlation was observed between the amount of lipase bound to the monolayer and the surface pressure increase, in agreement with previous studies. Conversely, with neutral lipid monolayers the amount of lipase bound to the monolayer was not found to be surface pressure dependent. This latter behaviour observed with lipases on neutral films is not specific to lipases, since it was also observed with bovine serum albumin and beta-lactoglobulin A. Lipase activity in the presence of various proteins was investigated with monomolecular films of glycerol didecanoate, either at constant surface area or at constant surface pressure. Depending upon the nature of the lipase and the protein, inhibition of lipase activity was either observed or not. Inhibition was correlated with a decrease in lipase surface concentration. The ability of the various proteins to inhibit lipolysis is: (i) a function of their excess versus lipase in the bulk phase, and: (ii) correlated with their penetration capacity (i.e., the initial rate of surface pressure increase of a glycerol didecanoate monolayer having an initial surface pressure of 20 dyn/cm, after the injection-of the protein). Since lipase inhibition was observed with low surface densities of inhibitory proteins, a long-range effect is probably involved in the mechanism of interfacial lipase inhibition. The nature of the ionic charge added to the monolayer by the protein is not critical for determining lipase adsorption or desorption. It is hypothesized that the lack of lipase adsorption to, or desorption from, the lipid monolayer results from a change in the organization of the hydrocarbon moiety of the lipid.     

Nanostructured nonionic thymidine nucleolipid self-assembly materials

Three nucleoside lipids have been synthesized: 3'-oleoylthymidine, 3',5'-dioleoylthymidine, and 3'-phytanoylthymidine. Differential scanning calorimetry and X-ray diffraction have been employed to characterize the physical properties of these neat lipids. Polarizing optical microscopy, small-angle X-ray scattering, and cryo-transmission electron microscopy techniques have been used to investigate the phase behavior in aqueous systems. Both oleoyl-based nucleoside lipids adopted a lamellar crystalline phase in the neat form at room temperature, and the phytanoyl derivative exhibited a fluid isotropic phase. Under excess water conditions, the presence of one branched (phytanoyl) or one unsaturated (oleoyl) chain promoted the formation of a liquid-crystalline lamellar phase at physiological temperatures. In contrast, the 3',5'-dioleoylthymidine derivative is nonswelling and does not exhibit lyotropic liquid-crystalline phase behavior. The nucleolipids' propensity for DNA-type binding and recognition has been evaluated by using a monolayer system to measure surface pressure-area isotherms in a Langmuir trough and indicates that the nucleoside base is available for nonspecific hydrogen bonding in the monolayer liquid expanded state for the single-chain nucleolipids but not for the dual-chain amphiphile.     

Mixed alkaline phosphatase/sphingomyelin monolayer at the air-buffer interface: phase behavior and morphology

The glycosylphosphatidyl inositol(GPI)-anchored proteins are localized on the outer of the plasma membrane and clustered in membrane microdomain known as lipid rafts. Among them, mammalian alkaline phosphatase(AP) is an enzyme widely distributed. So, it has important biological significance to study the combination of AP with lipid monolayer. In our work, the interaction between AP and sphingomyelin has been studied at the air-buffer interface as a biomimetic membrane system by the Langmuir film technique and atomic force microscopy. The surface pressure-area isotherm for the mixed alkaline phosphatase/sphingomyelin monolayer shown the presence of a transition from a liquid-expanded phase to the liquid-expanded/liquidcondensed coexist phase. And the surface compressional modulus suggested the mixed alkaline phosphatase/sphingomyelin monolayer has larger compressibility compared with the pure sphingomyelin monolayer. Besides, according to the micrographs, we inferred when combined with lipid monolayer at the air-buffer interface, the AP molecules formed polymer not multilayer or micelle. And, according to the limiting molecules area of AP, we inferred that 12 AP molecules formed a hexagon polymer unit.     

Progress in characterization of Langmuir monolayers by consideration of compressibility

Over decades, information about the rheological properties of the condensed monolayer phases has been obtained by introduction of a two-dimensional compressibility which is defined on the basis of the surface pressure-molecular area (Pi-A) features of the monolayer. Since the last decade, fundamental progress was attained in the experimental determination of the main characteristics of Langmuir monolayers in microscopic and molecular scale. Already smallest changes in the molecular structure of the amphiphile can result in changes in the molecular arrangement in the monolayer and thus, in changes of the main characteristics of the monolayer such as, the surface pressure-area per molecule (Pi-A) isotherms, the shape and texture of the condensed phase domains and the two-dimensional lattice structure. As the classical equations of state allowed only characterisation of the fluid (gaseous, liquid-expanded) state, thermodynamically based equations of state, which consider also the aggregation of the monolayer material to the condensed phase, have been developed. The present review focuses particularly to amphiphilic monolayers, the Pi-A isotherms of which indicate the existence of two condensed phases. For this case, the experimental results of the differences in the structure features and phase properties are discussed. The generalisation of the equation of state for Langmuir monolayers developed for the case that one, two or more phase transitions in the monolayer take place, is in agreement with the experimental results that the two-dimensional compressibility of the condensed phases undergoes a jump at the phase transition, whereas the compressibility is proportional to the surface pressure within one of the condensed phases. An example is presented which explains the procedure of the theoretical analysis of Pi-A isotherms indicating the existence of two condensed phases. An element of the procedure is the application of the general principle that the behaviour of any thermodynamic system is determined by the stability condition. An interesting anisotropy of the compressibility is revealed by GIXD studies of the S-phase of octadecanol monolayers. However, similar studies performed close to the LS-S-phase transition would result in a thermodynamically impossible negative compressibility. Close to this phase transition, the compressibility cannot be determined from the positions of the maxima because the monolayer is in a disordered state attributed to elastic distortions by fluctuations with the structure of the new phase in the surrounding matrix without destroying the quasi-long-range positional order.     

Phase behaviour of poly-(λ-benzyl-L-glutamate) in benzyl alcohol

Polarized optical microscopy has been used to investigate phase transitions in the poly-(λ-benzyl-L-glutamate) benzyl alcohol system and these have been compared with the predictions of Flory. All of the samples studied form gels at room temperature. The behaviour of the lowest concentration studied, 5 per cent by volume, shows transitions in the optical microscope compatible with the Flory phase diagram, becoming isotropic at elevated temperatures. Gels of higher concentrations exhibit bulk phase separation into an isotropic liquid phase and an anisotropic phase at room temperature, also in accord with the Flory predictions; the texture of the anisotropic phase varies with concentration. At higher temperatures these concentrations exhibit two coexistent anisotropic phases.     

Externally applied electric fields on immiscible lipid monolayers: repulsion between condensed domains precludes domain migration

Lipid and protein molecules anisotropically oriented at a hydrocarbon-aqueous interface configure a dynamic array of self-organized molecular dipoles. Electrostatic fields applied to lipid monolayers have been shown to induce in-plane migration of domains or phase separation in a homogeneous system. In this work, we have investigated the effect of externally applied electrostatic fields on different lipid monolayers exhibiting surface immiscibility. In the monolayers studied, lipids in the condensed state segregate in discontinuous round-shaped domains, with the lipid in the liquid-expanded state forming the continuous phase. The use of fluorescent probes with selective phase partitioning allows analyzing by epifluorescence microscopy the migrations of the domains under the influence of inhomogeneous electric fields applied to the surface. Our observations indicate that a positive potential applied to an electrode placed over the monolayer promotes a repulsion of the domains until a steady state is reached, indicating the presence of a force opposed to the externally applied electric force. The experimental results were modeled by considering that the opposing force is generated by the dipole-dipole repulsion between the domains.     

Phase transition of alkylsilane monolayers studied by temperature-dependent grazing incidence X-ray diffraction

The phase transition of organosilane monolayers on Si-wafer substrate surfaces prepared from octadecyltrichlorosilane (OTS) or docosyltrichlorosilane (DOTS) was investigated on the basis of grazing incidence X-ray diffraction (GIXD) at various temperatures. The OTS monolayer was prepared by a chemisorption method. The DOTS monolayer was prepared by a water-cast method (DOTS). The GIXD measurement clarified that the OTS monolayer also changed from hexagonal phase to amorphous state above a melting point of otadecyl groups. The GIXD measurements also clarified that the molecular aggregation state of the DOTS monolayer changes from an anisotropic phase to an isotropic phase with an increase in temperature. An estimated linear thermal expansion coefficient of the lattice lengths of a and b of the DOTS monolayer in the rectangular crystalline state assigned a similar value to those of bulk polyethylene with an orthorhombic crystalline lattice. The setting angle of the ab plane of the rectangular DOTS monolayer also showed similar behavior to that of the ab plane of bulk polyethylene.     

Phase Behavior of a Designed Cyclopropyl Analogue of Monoolein: Implications for Low‐Temperature Membrane Protein Crystallization

Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low‐temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small‐angle X‐ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high‐temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP‐based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 °C as well as 4 °C.     

Forces between cylindrical nanoparticles in a liquid crystal

Using classical density functional theory, the forces between two cylindrical nanoparticles in a liquid crystal solvent are calculated. Both the nematic and isotropic phases of the solvent are considered. In the nematic phase, the interaction is highly anisotropic. At short range, changes in the defect structure around the cylinders leads to a complex interaction between them. In the isotropic phase, an attractive interaction arises due to overlap between halos of ordered fluid adsorbed on the surfaces of the cylinders.     

Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.     

Globular and bicontinuous phases of nonionic surfactant films

Nonionic surfactants of the alkyloligoethylene oxide type form, with water and oil, a range of isotropic a liquid crystalline phases. We analyse the phase behaviour using the flexible surface model and argue that the strong temperature dependence is caused by the fact that the monolayer spontaneous curvature decreases strongly with increasing temperature. This is exemplified with the behaviour of bicontinuous microemulsions, showing a symmetric behaviour around the balanced state, globular microemulsions, behaving as hard spheres near the emulsification failure boundary, and sponge phases appearing when the monolayer spontaneous mean curvature is towards the abundant solvent. It is argued that there is a hierarchy of free energy contributions determining the preferred aggregate shape/phase. With a given oil-water ratio and a surfactant concentration that fixes the polar/apolar interfacial area, the most important free energy contribution comes from having a mean curvature close to the spontaneous curvature. The Gaussian curvature and the entropy terms become important when selecting between structures of similar mean curvature. At higher concentrations, surface forces and higher order elastic terms become significant.     

Fatty-acid monolayers at the nematic/water interface: phases and liquid-crystal alignment

The two-dimensional (2D) phases of fatty-acid monolayers (hexadecanoic, octadecanoic, eicosanoic, and docosanoic acids) have been studied at the interface of a nematic liquid crystal (LC) and water. When observed between crossed polarizers, the LC responds to monolayer structure owing to mesoscopic alignment of the LC by the adsorbed molecules. Similar to Langmuir monolayers at the air/water interface, the adsorbed monolayer at the nematic/water interface displays distinct thermodynamic phases. Observed are a 2D gas, isotropic liquid, and two condensed mesophases, each with a characteristic anchoring of the LC zenithal tilt and azimuth. By varying the monolayer temperature and surface concentration we observe reversible first-order phase transitions from vapor to liquid and from liquid to condensed. A temperature-dependent transition between two condensed phases appears to be a reversible swiveling transition in the tilt azimuth of the monolayer. Similar to monolayers at the air/water interface, the temperature of the gas/liquid/condensed triple-point temperature increased by about 10 degrees C for a two methylene group increase in chain length. However, the absolute value of the triple-point temperatures are depressed by about 40 degrees C compared to those of analogous monolayers at the air/water interface. We also observe a direct influence by the LC layer on the mesoscopic and macroscopic structure of the monolayer by analyzing the shapes and internal textures of gas domains in coexistence with a 2D liquid. An effective anisotropic line tension arises from elastic forces owing to deformation of the nematic director across phase boundaries. This results in the deformation of the domain from circular to elongated, with a distinct singularity. The LC elastic energy also gives rise to transition zones displaying mesoscopic realignment of the director tilt or azimuth between adjacent regions with a sudden change in anchoring.     

Interaction between puroindolines and the major polar lipids of wheat seed endosperm at the air-water interface

To understand the role of the puroindolines (PIN-a and PIN-b) in the defense mechanism and stabilization of lipid films in the gas cell of bread dough, we have isolated the proteins and lipids from wheat seed endosperm and studied their interaction at the air/water interface using a Langmuir trough. The nature and shape of the pressure–area compression isotherms of the lipid monolayer in the presence of puroindolines in the subphase depended on the concentration of protein. A distinct phase separation occurred, when the concentration of protein in the subphase increased. The interfacial elasticity of the lipid monolayer in the presence of puroindolines in the subphase was higher than the pure lipid. Injection of protein beneath the preexisting lipid monolayer resulted in the increase of surface pressure due to the penetration of proteins. The extent of penetration depended on the nature of lipid head groups as well as on the initial surface pressure. The penetration of puroindolines to lipid monolayer was observed to be zero after crossing a critical initial surface pressure. The magnitude of the critical initial surface pressure for anionic lipids was significantly higher than the zwitterionic and nonionic lipids. The experimental results showed that both PIN-a and PIN-b had more affinity for anionic polar lipids than the neutral polar lipids and stabilized the lipid monolayer.     

Aggregation of a peptide antibiotic alamethicin at the air-water interface and its influence on the viscoelasticity of phospholipid monolayers

The aggregation properties of an antibiotic membrane-active peptide alamethicin at the air-water interface have been studied using interfacial rheology and fluorescence microscopy techniques. Fluorescence microscopy of alamethicin monolayers revealed a coexistence of liquid expanded (LE) and solid phases at the surface concentrations studied. Interfacial oscillatory shear measurements on alamethicin monolayers indicate that its viscoelastic properties are determined by the area fraction of the solid domains. The role of zwitterionic phospholipids dioleoylphosphatidyl choline (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE) on the peptide aggregation behavior was also investigated. Fluorescence microscopy of alamethicin/phospholipid monolayers revealed an intermediate phase (I) in addition to the solid and LE phase. In mixed monolayers of phospholipid (L)/alamethicin (P), with increase in L/P, the monolayer transforms from a viscoelastic to a viscous fluid with the increase in area fraction of the intermediate phase. Further, a homogeneous mixing of alamethicin/lipid molecules is observed at L/P > 4. Our studies also confirm that the viscoelasticity of alamethicin/phospholipid monolayers is closely related to the alamethicin/phospholipid interactions at the air-water interface.     

Tethered bilayer lipid membranes self-assembled on mercury electrodes

In order to incorporate integral proteins in a functionally active state, metal-supported lipid bilayers must have a hydrophilic region interposed between the bilayer and the metal. This region is realized with a hydrophilic molecule terminating at one end with a sulfhydryl or disulfide group that anchors this "hydrophilic spacer" to the surface of a metal, such as gold or mercury. The other end of the hydrophilic spacer may be covalently linked to the polar head of a phospholipid molecule, giving rise to a supramolecule called "thiolipid" (TL). With respect to gold, mercury has the advantage of providing a defect-free and fluid surface to the self-assembling spacer. Hydrophilic spacers consisting of a polyethyleneoxy or a hexapeptide chain, as well as thiolipids derived from these spacers, were employed to fabricate mercury-supported lipid bilayers. The formation of a lipid bilayer on top of a self-assembled monolayer of a hydrophilic spacer, or of a single-lipid monolayer on top of a self-assembled monolayer of a thiolipid, was realized by simply immersing the coated mercury electrode into an aqueous solution across a lipid film previously spread on its surface at its spreading pressure. Particularly stable mercury-supported lipid bilayers were obtained by using thiolipids. The biomimetic properties of these lipid bilayers were tested by incorporating channel-forming polypeptides (gramicidin and melittin) and proteins (OmpF porin). The effect of the transmembrane potential on the function of these channels was estimated by using a simple electrostatic model of the mercury-solution interphase.     

甲基纤维素/二氯乙酸液晶溶液的相转变

本文研究了甲基纤维素/二氯乙酸液晶溶液液晶相与各向同性相之间相互转变的过程。在连续升温过程中,由于液晶相内部有序度的非均一性,升温速率对液晶相织构随温度的变化有较大的影响。在等温相转变过程中,无论是从各向同性态转变为液晶态,还是其逆过程,从液晶态转变为各向同性态,相转变曲线均具有指数方程特征。利用与 Avrami 方程相似的指数方程进行处理,在从各向同性态转变为液晶态时,指数 n 大部分约为1。但液晶相向各向同性相在低于 Tc 时的相转变中,n 均小于1。相转变速率受溶液浓度和温度的影响。在质量分数为0.259的浓度时,液晶相向各向同性相的转变在低于 Tc 时39.5℃进行得最快。