Calorimetric detection of curvature strain in phospholipid bilayers.

Phospholipids in biological membranes are arranged as bilayers. When constrained to pack into planar bilayers, certain phospholipids will form unstable structures as a consequence of their molecular shape and noncovalent bonding. This produces curvature strain which may provide energy for certain membrane processes. We demonstrate that an exothermic process associated with the relief of curvature strain can be detected calorimetrically. The enthalpy for the incorporation of a few percent lysophosphatidylcholine into large unilamellar vesicles of monomethyldioleoylphosphatidylethanolamine at pH 7.4 is exothermic but it is endothermic for stable bilayers such as this same lipid at pH 9 or dioleoylphosphatidylcholine at pH 7.4 or 9. The addition of lysophosphatidylcholine to monomethyldioleoylphosphatidylethanolamine at pH 7.4 is exothermic only for the addition of the first few percent of lysophosphatidylcholine and then it becomes endothermic. The size of the exothermic heat change is sensitive to changes in temperature, while the endothermic processes are relatively temperature-insensitive. The exothermic heat is also larger when 1 or 2 mol % of diolein is incorporated into vesicles of monomethyldioleoylphosphatidylethanolamine. These results are all consistent with the exothermic process corresponding to the relief of curvature strain in bilayers having a tendency to convert to the hexagonal phase. It provides a demonstration that considerable energy may be released upon the incorporation of certain molecules into membranes which have a low radius of spontaneous curvature.     

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the ²H NMR spectra of 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.     

Kinetic investigations on the phase transition of phospholipid bilayers

Pressure-jump experiments were performed on vesicles and liposomes of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine following the time course of solution turbidity. For both lipids two relaxation effects were evaluated the time constants of which exhibit clear maxima at the midpoint of the phase transition. The time constants lie for vesicles in the 100 μs and 1 ms ranges and for liposomes in the 1 ms and 10 ms ranges. The processes are slightly faster for dimyristoyl phosphatidylcholine than for dipalmitoyl phosphatidylcholine. All relaxation times are concentration-independent. The time constant and amplitude behaviours indicate that all processes are cooperative in agreement with previous interpretations. It is demonstrated that cooperative units can be evaluated from the relaxation amplitudes. These are of the same order of magnitude as those obtained from static experiments. On the grounds of the present kinetic investigation we can state that the application of the linear Ising model to two-dimensional processes as attempted for the static lipid phase transition is inadequate.     

Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.     

Hydration of phospholipid bilayers in the presence and absence of cholesterol

The number of water molecules bound (unfreezable) by a molecule of dipalmitoyl phosphatidylserine (DPPS) or by a molecule of dipalmitoyl phosphatidylcholine (DPPC) alone or in mixtures with cholesterol was determined by differential scanning calorimetry (DSC). When the phospholipids are in the gel state and in the absence of cholesterol, molecule of DPPS binds about 3.5 molecules of water and molecule of DPPC binds about 6 molecules of water. Number of water molecules bound increases when cholesterol crystallites are formed in the bilayer. For DPPS-cholesterol mixture at X(chol) -0.5, as well as for DPPC-cholesterol mixture at X(chol) -0.5 about 7 water molecules are bound.     

The range and shielding of dipole-dipole interactions in phospholipid bilayers

In molecular dynamics simulations of lipid bilayers, the structure is sensitive to the precise treatment of electrostatics. The dipole-dipole interactions between headgroup dipoles are not long-ranged, but the area per lipid and, through it, other properties of the bilayer are very sensitive to the detailed balance between the perpendicular and in-plane components of the headgroup dipoles. This is affected by the detailed properties of the cutoff scheme or if long-range interactions are included by Ewald or particle-mesh Ewald techniques. Interaction between the in-plane components of the headgroup dipoles is attractive and decays as the inverse sixth power of distance. The interaction is screened by the square of a dielectric permittivity close to the value for water. Interaction between the components perpendicular to the membrane plane is repulsive and decays as the inverse third power of distance. These interactions are screened by a dielectric permittivity of the order 10. Thus, despite the perpendicular components being much smaller in magnitude than the in-plane components, they will dominate the interaction energies at large distances.     

Competing interactions contributing to alpha-helical stability in aqueous solution.

The stability of a 15-residue peptide has been investigated using CD spectroscopy and molecular simulation techniques. The sequence of the peptide was designed to include key features that are known to stabilize alpha-helices, including ion pairs, helix dipole capping, peptide bond capping, and aromatic interactions. The degree of helicity has been determined experimentally by CD in three solvents (aqueous buffer, methanol, and trifluoroethanol) and at two temperatures. Simulations of the peptide in the aqueous system have been performed over 500 ps at the same two temperatures using a fully explicit solvent model. Consistent with the CD data, the degree of helicity is decreased at the higher temperature. Our analysis of the simulation results has focused on competition between different side-chain/side-chain and side-chain/main-chain interactions, which can, in principle, stabilize the helix. The unfolding in aqueous solution occurs at the amino terminus because the side-chain interactions are insufficient to stabilize both the helix dipole and the peptide hydrogen bonds. Loss of capping of the peptide backbone leads to water insertion within the first peptide hydrogen bond and hence unfolding. In contrast, the carboxy terminus of the alpha-helix is stable in both simulations because the C-terminal lysine residue stabilizes the helix dipole, but at the expense of an ion pair.     

Calorimetric studies of the interactions of guanidinium hydrochloride and potassium iodide with model amides in aqueous solution.

The enthalpies of transfer, ΔH_tr, of a series of amides from water to aqueous solutions of either guanidinium hydrochloride (GuHCl) or potassium iodide were obtained from calorimetric measurements at 25°C. The amides were studied at molalities around 10^?2 m while salt molalities ranged from 0–10 m. The amides investigated were Ac-Gly-NHMe, Ac-Gly-Gly-NHMe, Ac-Ala-NHMe, and Ac-Leu-NHMe. Use of an additivity assumption allowed the calculation of group contributions to ΔH_tr in these two salt systems for the methyl group, leucyl side chain, and the peptide backbone unit. Values of the entropy of transfer were also obtained. The great ability of GuHCl to randomize protein structures appears to arise from effects on polar and nonpolar groups, which are characterized by enthalpies and entropies of transfer not substantially different from those with KI, a salt comprised of ions of comparable size and polarizability. The difference in the sign of the free energies of transfer of nonpolar groups from water to MX solutions, negative for GuHCl and positive for KI, is the result of these small differences in enthalpies and entropies of transfer. Variations in water structure produced by differences in ionic properties rather than a mode of action for GuHCl very different from that of other salts characterizes its superior denaturing ability.     

Thermal stability of beta-lactoglobulin in the presence of aqueous solution of alcohols and polyols

A systematic study concerning the effect of aqueous solution of alcohols and polyols with four carbon atoms on β-lactoglobulin stability is presented. The protein was chosen due to its functional properties and applications in food and pharmaceutical industries and because its structure and properties in aqueous solution have been widely described. The alcohols having a four carbon chain were selected to examine the effect of the gradual increase in the number of OH groups on protein stability.

Protein thermal stability in water, buffers and dilute aqueous solutions of 1-butanol, 1,2-butanediol, 1,2,4-butanetriol and 1,2,3,4-butanetetrol was evaluated by fluorescence spectroscopy. The results were used to determine the temperature range in which the unfolding process is reversible and the protein denaturation temperature in acetate buffer pH 5.5 and in the aqueous mixed solvents. Thermodynamic results show that alcohol denaturating effect diminishes gradually as the number of OH groups increase.     

NMR investigations of interactions between anesthetics and lipid bilayers

Interactions between anesthetics (lidocaine and short chain alcohols) and lipid membranes formed by dimyristoylphosphatidylcholine (DMPC) were studied using NMR spectroscopy. The orientational order of lidocaine was investigated using deuterium NMR on a selectively labelled compound whereas segmental ordering in the lipids was probed by two-dimensional ¹H-¹³C separated local field experiments under magic-angle spinning conditions. In addition, trajectories generated in molecular dynamics (MD) computer simulations were used for interpretation of the experimental results. Separate simulations were carried out with charged and uncharged lidocaine molecules. Reasonable agreement between experimental dipolar interactions and the calculated counterparts was observed. Our results clearly show that charged lidocaine affects significantly the lipid headgroup. In particular the ordering of the lipids is increased accompanied by drastic changes in the orientation of the P-N vector in the choline group.     

Lipid-protein interactions of the human erythrocyte concanavalin a receptor in phospholipid bilayers

The interaction of the human erythrocyte concanavalin A receptor (a subpopulation of Band 3) with phospholipids has been investigated using differential scanning microcalorimetry of reconstituted vesicles prepared by detergent dialysis. The mean diameter of dialyzed phospholipid vesicles jumps dramatically on inclusion of the concanavalin A receptor and then increases linearly with the fraction of protein in the bilayer. The glycoprotein has a dramatic effect on the phospholipid gel to liquid-crystalline phase transition, and ΔH decreases linearly with increasing mole fraction of protein up to a protein/lipid mole ratio of around 1:1160. Extrapolation of this data indicates that each concanavalin A receptor is able to perturb about 685 molecules of dimyristoylphosphatidylcholine, withdrawing them from the main phase transition. The cooperativity of phospholipid melting is profoundly disrupted by small amounts of glycoprotein, with the cooperative unit dropping to less than half its initial values at a protein/lipid mole ratio of 1:3800. A break occurs in the ΔH curve as the protein/lipid mole ratio is increased above 1:1160, and ΔH then increases linearly with increasing amounts of concanavalin A receptor in the bilayer. This phenomenon may be interpreted in terms of protein-protein aggregation which occurs in the phospholipid bilayer above a certain critical mole fraction of concanavalin A receptor, resulting in perturbed phospholipids being returned to the phase transition. In addition, the hydrophilic domains of the glycoprotein may exist in two different conformations depending on the protein concentration in the bilayer, and these may differ in their ability to interact with phospholipid headgroups at the membrane surface.     

Geometry of phase-separated domains in phospholipid bilayers by diffraction-contrast electron microscopy.

The sizes and shapes of solidus (gel) phase domains in the hydrated molecular bilayers of dilauroylphosphatidylcholine/dipalmitoylphasphatidylcholine (DLPC/DPPC) (1:1) and phosphatidylserine (PS)/DPPC (1:2) are visualized directly by low dose diffraction-contrast electron microscopy. The temperature and humidity of the bilayers are controlled by an environmental chamber set in an electron microscope. The contrast between crystalline domains is enhanced by electron optical filtering of the diffraction patterns of the bilayers. The domains are seen as a patchwork in the plane of the bilayer, with an average width of 0.2-0.5 micrometer. The percentage of solidus area measured from diffraction-contrast micrographs at various temperatures agrees in general with those depicted by known phase diagrams. The shape and size of the domains resemble those seen by freeze-fracture in multilamellar vesicles. Temperature-related changes in domain size and in phase boundary per unit area are more pronounced in the less miscible DLPC/DPPC mixture. No significant change in these geometric parameters with temperature is found in the PS/DPPC mixture. Mapping domains by their molecular diffraction signals not only verifies the existance of areas of different molecular packing during phase separation but also provides a quantitative measurement of structural boundaries and defects in lipid bilayers.     

Calorimetric,volumetric and structural studies of the interaction between chlorogenic acid and dipalmitoylphosphatidylcholine bilayers

Chlorogenic acid (CGA) is the main component of coffee and an antioxidant. CGA has been reported to bear various good health effects. At the same time, it has been found that the addition of CGA induces an undesirable deformation of red blood cells. This fact suggests that CGA may bind to the proteins or/and membrane lipids of red blood cells. This study aimed to examine how CGA binds the bilayers of phosphatidylcholine (PC), one of red blood cells' primary lipids. To this end, we investigated the effect of CGA on the phase behavior and the structure of dipalmitoyl-PC (DPPC) bilayers in the form of multi-lamellar vesicles. Calorimetry and dilatometry measurements showed that the DPPC chain melting transition cooperativity decreases as increasing CGA concentrations. In addition, X-ray diffraction results showed that the lamellar repeat periodicity becomes disordered, and the periodicity disappears completely at high CGA concentrations. Together with these findings, it can be inferred that the CGA molecules do not penetrate inside the DPPC bilayers but bind to their surface in a negatively charged form.     

Influence of proteins on the reorganization of phospholipid bilayers into large domains.

Using large (5-10 microns) vesicles formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera, and digital image processor, we examined the effects of membrane proteins on phospholipid domain formation. In vesicles composed of phosphatidic acid and phosphatidylcholine, incubation with cytochrome c induced the reorganization of phospholipids into large phosphatidic acid-enriched domains with the exclusion of phosphatidylcholine. Cytochrome c binding was demonstrated to be highest in the phosphatidic acid-enriched domain of the vesicle using the absorbance of the heme moiety for visualization. Both binding of cytochrome c and phospholipid reorganization were blocked by pretreatment of the vesicles with 0.1 M NaCl. The pore forming peptide gramicidin was examined for the effects of an integral protein on domain formation. Initially, gramicidin distributed randomly within the vesicle and showed no phospholipid specificity. Phosphatidic acid domain formation in the presence of 2.0 mM CaCl2 or 100 microM cytochrome c was not affected by the presence of 5 mol % gramicidin within the vesicles. In both cases, gramicidin was preferentially excluded from the phosphatidic acid-enriched domain and became associated with phosphatidylcholine-enriched areas of the vesicle. Thus, cytochrome c caused a major reorganization of both the phospholipids and the proteins in the bilayer.     

Molecular simulation study of phospholipid bilayers and insights of the interactions with disaccharides

Molecular simulations of hydrated dipalmitoylphosphatidylcholine lipid bilayers have been performed for temperatures in the range of 250-450 K. The area per headgroup increases with temperature from 58 to 77 A(2). Other properties such as hydration number, alkyl tail order parameter, diffusion coefficients, and radial distribution functions exhibit a clear dependence on temperature. Simulations of bilayers have also been performed in the presence of two disaccharides, namely trehalose and sucrose, at concentrations of up to 18 wt % (lipid-free basis). The simulated area per headgroup of the bilayer is not affected by the presence of the disaccharides, suggesting that the overall structure of the bilayer remains undisturbed. The results of simulations reveal that the interaction of disaccharide molecules with the bilayer occurs at the surface of the bilayer, and it is governed by the formation of multiple hydrogen bonds to specific groups of the lipid. Disaccharide molecules are observed to adopt specific conformations to fit onto the surface topology of the bilayer, often interacting with up to three different lipids simultaneously. At high disaccharide concentrations, the results of simulations indicate that disaccharides can serve as an effective replacement for water under anhydrous conditions, which helps explain their effectiveness as lyophilization agents for liposomes and cells.     

Lateral interactions among phosphatidylcholine and phosphatidylethanolamine head groups in phospholipid monolayers and bilayers

We develop theory for the lateral interactions among the zwitterionic head groups of phospholipids in monolayers and bilayers, particularly phosphatidylcholine (PC) and phosphatidylethanolamine (PE). With the P- end of the head group anchored at the water/hydrocarbon interface, a balance of two effects dictates the angle that the P--N+ dipole makes with respect to the plane of the bilayer: N+ is driven toward water due to the (Born) electrostatic free energy, but the hydrophobic effect drives the methyl and methylene groups around the N+ charge toward the hydrocarbon. The only adjustable parameter of the model is the average fluctuation of the oil/water interface or, alternatively, the dielectric constant of the hydrocarbon phase. The model predicts that at 5 degrees C the head group dipole should lie largely in the bilayer plane, in accord with X-ray, neutron diffraction, and NMR studies. The theory makes the novel prediction that the N+ end of the dipole becomes increasingly submerged in hydrocarbon with increasing temperature, leading to strongly enhanced lateral repulsion between PC head groups. This prediction is in good agreement with second and third viral coefficients of monolayer lateral pressures, and with the temperature dependence of the former. The theoretical model is consistent with head group fluctuations measured by neutron diffraction of PC and PE bilayers. Because PE has a smaller hydrophobic cluster near N+, its lateral repulsion should be much smaller and less temperature dependent than for PC, also in agreement with equation-of-state measurements. This suggests why at high density PE monolayers have higher melting temperatures than PC monolayers and more propensity for reversed curvature.     

Study of hydrophobic interactions between acylated proteins and phospholipid bilayers using BIACORE

Intracellular proteins of eukaryotic cells are frequently covalently modified by the addition of long chain fatty acids. These modifications are thought to allow otherwise soluble proteins to associate with membranes by lipid-lipid based hydrophobic interactions. The purpose of this work was to quantify the effect of acyl chain length on hydrophobic interactions between acylated proteins and phospholipid monolayers. The binding of an artificially acylated model protein to electrically neutral phospholipids was studied by surface plasmon resonance, using BIACORE. Kinetic rates for the binding of bovine pancreatic ribonuclease A (RNase A), monoacylated on its N-terminal lysine with fatty acids of 10, 12, 14, 16 or 18 carbon atoms, to phospholipids on hydrophobic sensor chips, were measured. Unlike unmodified ribonuclease, acylated RNase A bound to the phospholipids, and the association level increased with the acyl chain length to reach a maximum for C16. Reproducible kinetics were obtained which did not fit a 1:1 Langmuir model but rather a two-step binding profile.     

Interactions of colicin A domains with phospholipid monolayers and liposomes: relevance to the mechanism of action

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).     

Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains

The stability and structure of human apolipoprotein (apo) E3 in aqueous solution were investigated by guanidine HCl denaturation and limited proteolysis. The guanidine HCl denaturation curve, as monitored by circular dichroism spectroscopy, was biphasic; the two transition midpoints occurred at 0.7 and 2.5 M guanidine HCl, indicating that there are stable intermediate structures in the unfolding of apoE. Limited proteolysis of apoE with five enzymes demonstrated two proteolytically resistant regions, an amino-terminal domain (residues 20-165) and a carboxyl-terminal domain (residues 225-299). The region between them was highly susceptible to proteolytic cleavage. Because of their similarity to the proteolytically resistant regions, the amino-terminal (residues 1-191) and carboxyl-terminal (residues 216-299) thrombolytic fragments of apoE were used as models for the two domains. Guanidine HCl denaturation of the carboxyl- and amino-terminal fragments gave transition midpoints of 0.7 and 2.4 M guanidine HCl, respectively. The results establish that the two domains identified by limited proteolysis correspond to the two domains detected by protein denaturation experiments. Therefore, the thrombolytic fragments are useful models for the two domains. The free energies of denaturation calculated from the denaturation curves of intact apoE or the model domains were approximately 4 and 8-12 kcal/mol for the carboxyl- and amino-terminal domains, respectively. The value for the carboxyl-terminal domain is similar to those of previously characterized apolipoproteins, whereas the value for the amino-terminal domain is considerably higher and resembles those of soluble globular proteins. These studies suggest that, in aqueous solution, apoE is unlike other apolipoproteins in that it contains two independently folded structural domains of markedly different stabilities: an amino-terminal domain and a carboxyl-terminal domain, separated by residues that may act as a hinge region.     

Synergistic interactions between aqueous and membrane domains of a designed protein determine its fold and stability

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.