Structure Of Lipid Aggregates Research Articles

Use of attenuated total reflectance Fourier transform infrared spectroscopy to monitor the development of lipid aggregate structures

Attenuated total reflectance Fourier transform infrared spectroscopy is used to monitor the adsorption of 100 nm 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipid vesicles to the surfaces of Ge, electrolessly deposited Au, and a well formed self-assembled monolayer of 1-octadecanethiol. The interaction of DPPC vesicles in solution with these different surfaces yields distinctly different surface structures: intact DPPC vesicles on Ge, a supported phospholipid bilayer on an electrolessly deposited Au surface, and a phospholipid monolayer onto the hydrophobic self-assembled monolayer. IR peak position, bandwidth, and intensity are used to confirm structure formation and quantitation of the amount of lipid that desorbs during film formation.

Lipids, curvature, and nano‐medicine

The physical properties of the lamellar lipid-bilayer component of biological membranes are controlled by a host of thermodynamic forces leading to overall tensionless bilayers with a conspicuous lateral pressure profile and build-in curvature-stress instabilities that may be released locally or globally in terms of morphological changes. In particular, the average molecular shape and the propensity of the different lipid and protein species for forming non-lamellar and curved structures are a source of structural transitions and control of biological function. The effects of different lipids, sterols, and proteins on membrane structure are discussed and it is shown how one can take advantage of the curvature-stress modulations brought about by specific molecular agents, such as fatty acids, lysolipids, and other amphiphilic solutes, to construct intelligent drug-delivery systems that function by enzymatic triggering via curvature.Practical applications: The simple concept of lipid molecular shape and how it impacts on the structure of lipid aggregates, in particular the curvature and curvature stress in lipid bilayers and liposomes, can be exploited to construct liposome-based drug-delivery systems, e.g., for use as nano-medicine in cancer therapy. Non-lamellar-forming lysolipids and fatty acids, some of which may be designed to be prodrugs, can be created by phospholipase action in diseased tissues thereby providing for targeted drug release and proliferation of molecular entities with conical shape that break down the permeability barrier of the target cells and may hence enhance efficacy.

Electrostatic Bending of Lipid Membranes: How Are Lipid and Electrostatic Properties Interrelated?

Electrostatic modification of lipid headgroups and its effect on membrane curvature are not only relevant in a variety of contexts such as cell shape transformation and membrane tubulation but also are intriguingly implicated in membrane functions. For instance, the gating (open vs closed) properties of mechanosensitive channels can be influenced by membrane curvature and ion valence. However, a full theoretical description of membrane electrostatics is still lacking; in the past, membrane bending has often been considered under a few assumptions about how bending modifies lipid arrangements and surface charges. Here, we present a unified theoretical approach to spontaneous membrane curvature, C(0), in which lipid properties (e.g., packing shape) and electrostatic effects are self-consistently integrated. For the description of electrostatic interactions, especially between a lipid charge and a divalent counterion, we implement the Poisson-Boltzmann (PB) approach by incorporation of finite ionic sizes, so as to capture both lateral and transverse charge correlations on the membrane surface. Our results show that C(0) is sensitive to the way lipid rearrangements and divalent counterions are modeled. Interestingly, it can change its sign in the presence of divalent counterions, thus stabilizing reverse hexagonal (H(II)) phases. Our results show how electrostatic modification of headgroups influences the preferred structure of lipid aggregates (inverted micelles vs bilayers).

Structural effects caused by spray‐ and freeze‐drying of liposomes and bilayer disks

Cryo‐TEM and dynamic light scattering was used to investigate morphological changes induced by spray‐ and freeze‐drying of liposomes and nanosized bilayer disks composed of 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC), cholesterol, and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol)‐5000] (DSPE‐PEG) from lactose solution. Particular focus was put on the identification of structural alterations that risk influencing the performance of liposomes and bilayer disks as carriers for protein and peptide drugs. Significant changes in the lipid aggregate structure and/or size was noted upon dehydration. Uni‐lamellar liposomes tended to shrink in size and become bi‐lamellar as a consequence of the drying process. The same transformation was observed upon deliberate establishment of a lactose gradient over the membranes of liposomes in solution. A mechanism based on an osmotically driven invagination of the liposomes is proposed to explain the change from uni‐ to bi‐lamellar structures. PEGylation promoted formation of larger liposomes during spray‐drying, and had a similar, but less pronounced, effect also during freeze‐drying. The observed structural changes may have important consequences for the bioavailability of protein/peptide drugs bound to, or embedded in, the liposome membranes. The radius of bilayer disks increased upon both spray‐ and freeze‐drying, but the drying procedure did not change the open single‐bilayer structure of the disks. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2032–2048, 2010

Computation of Lipid Headgroup Interactions

The equilibrium structure of lipid aggregates is determined by the balance of numerous forces between hydrophobic acyl chains, hydrophilic lipid headgroups, and the lipid's environment. Among these forces, lipid headgroup interactions are both important to the stability of lipid structures and responsible for many of the interactions between biological membranes and aqueous solutes including ions and soluble peptides. In order to model these headgroup interactions, we consider the electrical properties of the headgroup molecules via the multipole expansion.

Interactions of phenytoin with lipids in mixed Langmuir monolayers

The behaviour of dipalmitoyl phosphatidylcholine (DPPC)/cholesterol monolayers on pure water and on water with phenytoin was studied by performing Langmuir monolayer technique experiments. Surface pressure–area isotherms were measured for pure DPPC, cholesterol films and their mixtures. The changes of the limiting molecular area, collapse pressure and maximum compressional modulus show that phenytoin can modify the structure of lipid aggregates. The introduction of phenytoin to the subphase reduces the deviations of mean molecular areas from additivity rule observed for mixed DPPC/cholesterol monolayers. Thermodynamic stability of the mixed monolayers, as compared to that of pure monolayers, was determined by analysis of the excess Gibbs free energy of mixing. Results indicate an increase of thermodynamic stability with increasing surface pressure and decrease of thermodynamic stability in the presence of phenytoin in the system. The existence of a minimum indicates that the mixed monolayers are thermodynamically more stable than the monolayers with separation between individual components, especially around x DPPC = 0.6, on both subphases.

Interaction of cytochrome c with cubic monoolein mesophases at limited hydration conditions: The effects of concentration, temperature and pressure

Incorporation of the protein cytochrome c (cyt c) into hydrated cubic mesophases of monoolein (MO) was investigated within a wide range of concentrations, temperatures and pressures by small-angle X-ray scattering (SAXS) and Fourier-transform infrared (FT-IR) spectroscopy. The SAXS data allow the determination of the topology of the mesophase and phase behavior of the lipid system. Further information on the lipid conformational states and hydrational properties were obtained using FT-IR spectroscopy. The effect of the lipid matrix on the structure and stability of the embedded cyt c was also studied by FT-IR spectroscopy by monitoring changes in secondary structural properties of the protein by analysis of the FT-IR amide I′ band. Incorporation of cyt c into the cubic phase of MO has significant effects on the structure and pressure stability of the system and the lipid bilayer and protein molecules undergo interrelated changes in their structural properties, even though the lipids are uncharged. Entrapping of low concentrations of cyt c (< 0.2 wt%) leads to changes only in the phase transition temperatures and pressures. In contrast, higher protein concentrations have a drastic effect on the molecular properties of the lipid system. Lipid–protein interactions lead to an increase in interfacial curvature which promotes the formation of a new, possibly partially micellar, cubic phase of crystallographic space group P4332 (Q212). It is very likely that the protein molecules are accommodated in the aqueous interior of inverted micellar aggregates in this structure. The IR data reveal that the new lipid aggregate structure is accompanied by an increase of carbonyl headgroup hydration and a decrease of lipid chain order. As a consequence of attractive protein–headgroup interactions, the pressure stability of this phase increases with increasing the protein content. Above ca. 4 kbar, ordered lamellar phases are induced which have the smallest partial molar volume. The unfolding temperature of cyt c is reduced by ca. 4 °C by incorporation of the protein into the lipid mesophases, as compared to the protein dissolved in water. Contrary to what would be expected for a pure confinement effect, which would lead to a significant increase of the unfolding temperature of the protein, our data clearly show that confinement effects can easily be overcompensated by attractive protein–lipid interactions. The latter lead to a slight destabilization of the protein. No pressure-induced unfolding of the protein occurs up to 16 kbar.

Fluorescence Probe Study of Bicelle Structure as a Function of Temperature: Developing a Practical Bicelle Structure Model

Bilayered mixed micelles or bicelles are magnetically anisotropic, self-assembling model membrane structures comprised of long-chain phospholipids and short-chain detergent molecules. In the most widely accepted model of this system, the bicelle is discoid in shape, with the short-chain DHPC molecules aggregating to form rims around long-chain DMPC bilayers. While this model is consistent with most NMR and scattering data (X-ray and neutron), it inadequately describes the liquid-crystalline behavior of bicelle solutions at temperatures where magnetic alignment occurs. Temperature plays a central role in the structure of lipid aggregates, and the impact of temperature on bicelles has not been studied as extensively as composition and concentration. Therefore, a series of fluorescence probe and resonance energy transfer (FRET) measurements of labeled bicelle solutions as a function of temperature were conducted to monitor lipid mixing as an indication of bicelle structure and aggregation. The results of the...

Effect of Lipase on Monoolein-Based Cubic Phase Dispersion (Cubosomes) and Vesicles

The effect of adding a Thermomyces (formerly Humicola) lanuginosa lipase (TLL) to a cubic dispersion of the binary monoolein (MO)-(H2O)-H-2 system and to vesicles formed by the ternary MO-sodium oleate (NaO)(H2O)-H-2 system at high water content has been studied. The change in lipid aggregate structure during the lipolytic process was observed by light microscopy, cryo-TEM, and dynamic light scattering, and the lipid composition was determined with HPLC. The sequence of lipid aggregate structures observed upon lipase addition to the cubic dispersion is cubic dispersion --> reversed cubic, hexagonal, and lamellar structures --> large polygonal structures --> dispersed oil droplets. The addition of lipase to MO/NaO aqueous-based vesicles did not change the morphology significantly. However, close to steady state, the vesicles changed from having smooth interfaces to irregular ones. When lipolysis reached steady state, the MO/(OA+NaO) ratio was similar in the two systems studied, although the morphology of the structures formed during lipolysis differed significantly. These differences are discussed in relation to the degree of dissociation of the fatty acid. (Less)

Domains of surfactant protein A that are required for lipid aggregate structures and surface active properties of surfactant.

Domains of surfactant protein A that are required for lipid aggregate structures and surface active properties of surfactant.

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles

The activity of phosphatidylserine (PS) synthase (CDP-1,2-diacyl- sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8.8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.

The Teaching of Biochemistry: An Innovative Course Sequence Based on the Logic of Chemistry

An innovative course sequence for the teaching of biochemistry is offered, which more truly reflects the common philosophy found in biochemistry texts: that the foundation of biological phenomena can best be understood through the logic of chemistry. Topic order is chosen to develop an emerging understanding that is based on chemical principles. Preeminent biological questions serve as a framework for the course. Lipid and lipid-aggregate structures are introduced first, since it is more logical to discuss the intermolecular association of simple amphiphiles to form micelle and bilayer formations than to discuss the complexities of protein structure/folding. Protein, nucleic acid, and carbohydrate structures are studied next. Binding, a noncovalent process and the simplest expression of macromolecular function, follows. The physical (noncovalent) transport of solute molecules across a biological membrane is studied next, followed by the chemical transformation of substrates by enzymes. These are logical extensions of the expression of molecular function, first involving a simpler (physical transport) and second, a more complex (covalent transformation) process. The final sequence involves energy and signal transduction. This unique course sequence emerges naturally when chemical logic is used as an organizing paradigm for structuring a biochemistry course. Traditional order, which seems to reflect historic trends in research, or even an order derived from the central dogma of biology can not provide this logical framework.

The relationship between compositional phase separation and vesicle morphology: Implications for the regulation of phospholipase A 2 by membrane structure

The action of phospholipase A 2 (PLA 2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA 2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.

Interconversions of lipid aggregates in rat and model bile

The dynamic interchange of cholesterol and the phase transition between nonmicellar and micellar aggregates in rat and model bile were characterized with gel-permeation chromatography, quasi-elastic light scattering, turbidity measurements, and by radiolabeling lipid aggregates in bile. Cholesterol partitioned into either the micellar or nonmicellar phases independent of the lipid aggregate structure. In model bile, increasing bile salt concentrations led to a decrease in the relative proportion of nonmicellar aggregates beginning at 5 mM taurocholate (TC), while the relative cholesterol content of the nonmicellar fraction increased from 1.0 to 2.7 +/- 2.0 (means +/- SD). In rats, creation of a biliary fistula resulted in a decrease of bile salts from 41 to 4 mM. Mixed micelles increased from 25 to 120 A in radius, while nonmicellar aggregates increased from 180 to 800 A in radius. Addition of TC to model bile (cholesterol:lecithin = 1:1) vesicles with total lipid concentrations less than 7 mM yielded a progressive shift of vesicles (450 A) to mixed micelles (30 A). For mixtures with higher total lipid concentrations, addition of TC promoted substantial vesicle aggregation and resulted in formation of a third phase containing lipid aggregates larger in size than the initial vesicles. These results suggest that rapid exchange of cholesterol occurs in bile and that significant remodeling of vesicles can occur. These alterations in vesicles include both enrichment in cholesterol content and formation of larger aggregates during increases in bile salt concentration.

Structure and dynamics of a glyceroglycolipid: a deuterium NMR study of head group orientation, ordering, and effect on lipid aggregate structure

The head group orientation and the motional characteristics of 1,2-di-O-tetradecyl-3-O-..beta..-D-glucopyranosylglycerol selectively /sup 2/H-labeled on the glucose moiety have been investigated by differential scanning calorimetry and /sup 2/H NMR. The glycolipid undergoes a major endothermic transition at 52/sup 0/C, which is attributed to the gel to liquid-crystal phase transition. The nature of a less energetic endothermic transition at 58/sup 0/C, determined to be a lamellar to hexagonal mesophase transition by /sup 2/H NMR, is confirmed by x-ray diffraction. In the Lamellar phase, the glycolipid head group undergoes axially symmetric motion and has an orientational order parameter S/sub mol/ of 0.45. The head group is extended away from the bilayer surface. On entering the hexagonal mesophase, the orientational order parameter for the sugar ring is reduced slightly to 0.38, but the local rotation axis undergoes a large reorientation with respect to the carbohydrate ring. In a phospholipid matrix, the orientation of the carbohydrate head group of the glycolipid is affected by the greater extension of the surface residues of the host lipid.