Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure

Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16–21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein–lipid interactions: the DYS R16–21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles

The thermodynamics of binding of the antibacterial peptide magainin 2 amide (M2a) to negatively charged small (SUVs) and large (LUVs) unilamellar vesicles has been studied with isothermal titration calorimetry (ITC) and CD spectroscopy at 45 degrees C. The binding isotherms as well as the ability of the peptide to permeabilize membranes were found to be qualitatively and quantitatively similar for both model membranes. The binding isotherms could be described with a surface partition equilibrium where the surface concentration of the peptide immediately above the plane of binding was calculated with the Gouy-Chapman theory. The standard free energy of binding was deltaG0 approximately -22 kJ/mol and was almost identical for LUVs and SUVs. However, the standard enthalpy and entropy of binding were distinctly higher for LUVs (deltaH0 = -15.1 kJ/mol, deltaS0 = 24.7 J/molK) than for SUVs (deltaH0 = -38.5 kJ/mol, deltaS0 = -55.3 J/molK). This enthalpy-entropy compensation mechanism is explained by differences in the lipid packing. The cohesive forces between lipid molecules are larger in well-packed LUVs and incorporation of M2a leads to a stronger disruption of cohesive forces and to a larger increase in the lipid flexibility than peptide incorporation into the more disordered SUVs. At 45 degrees C the peptide easily translocates from the outer to the inner monolayer as judged from the simulation of the ITC curves.     

Thermodynamics of the coil-alpha-helix transition of amphipathic peptides in a membrane environment: the role of vesicle curvature

The binding of peptides or proteins to a bilayer membrane is often coupled with a random coil-->alpha-helix transition. Knowledge of the energetics of this membrane-induced folding event is essential for the understanding of the mechanism of membrane activity. In a recent study [Wieprecht et al., J. Mol. Biol. 294 (1999) 785-794], we have developed an approach which allows an analysis of the energetics of membrane-induced folding. We have systematically varied the helix content of the amphipathic peptide magainin-2-amide by synthesizing analogs where two adjacent amino acid residues were substituted by their corresponding D-enantiomers and have measured their binding to small unilamellar vesicles (SUVs). Correlation of the binding parameters with the helicities allowed the evaluation of the thermodynamic parameters of helix formation. Since SUVs (30 nm in diameter) are characterized by a non-ideal lipid packing due to their high membrane curvature, we have now extended our studies to large unilamellar vesicles (LUVs) (100 nm in diameter) with a lipid packing close to planar membranes. While the free energy of binding was similar for SUVs and LUVs, the binding enthalpies and entropies were distinctly different for the two membrane systems. The thermodynamic parameters of the coil-helix transition were nevertheless not affected by the vesicle size. Helix formation at the membrane surface of LUVs (SUVs) was characterized by an enthalpy change of -0.8 (-0.7) kcal/mol per residue, an entropy change of-2.3 (-1.9) cal/mol K per residue, and a free energy change of -0.12 (-0.14) kcal/mol per residue. Helix formation accounted for approximately 50% of the free energy of binding underlining its major role as a driving force for membrane-binding.     

Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

Association of a protein with membrane vesicles at the collisional limit: studies with blood coagulation factor Va light chain also suggest major differences between small and large unilamellar vesicles

Vesicle size can be a very sensitive modulator of protein-membrane association. In addition, reactions at the collisional limit may be characteristic of many types of protein-membrane or protein-receptor interactions. To probe these effects quantitatively, we analyzed the association of blood clotting factor Va light chain (Va-LC) with phospholipid vesicles of 15-150-nm radius. The number of protein binding sites per vesicle was approximately proportional to vesicle surface area. Association rates approached the collisional limit, and the activation energy for the association reaction was 4.5 +/- 0.5 kcal/mol. In agreement with diffusional theory for this type of interaction at the collisional limit, the observed association rate constant for filling all sites was approximately proportional to the inverse of vesicle radius. This general property has important implications for many systems such as blood coagulation including possible slower association rates and higher Km values for reactions involving whole cells relative to those obtained for phospholipid vesicles. Dissociation rate constants for reactions that are near the collisional limit should also be proportional to the inverse of vesicle size if diffusional parameters are the only factors influencing dissociation. However, Va-LC bound to small unilamellar vesicles (SUVs, less than or equal to 15-nm radius) gave slower dissociation rates than Va-LC bound to large unilamellar vesicles (LUVs, greater than or equal to 35-nm radius). This indicated a change in KI, the intrinsic protein-phospholipid affinity constant for LUVs vs SUVs. The cumulative effect of association and dissociation rates resulted in higher affinity of Va-LC for SUVs than LUVs under equilibrium conditions. The latter was corroborated by competition binding studies. Furthermore, the temperature dependence of both rate constants indicated an entirely entropy-driven binding to LUVs but a largely enthalpy-driven binding to SUVs. Interactions which are largely entropic are thought to be ionic in nature. The differences observed between binding to LUVs and SUVs may reflect thermodynamic differences between these types of phospholipid structures.     

The use of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipids in determining transmembrane lipid distribution

Transbilayer lipid distribution of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was measured using ³¹P-nuclear magnetic resonance (NMR) spectroscopy, chemical modification with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dithionite reduction of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipid (NBD-lipid). The dithionite assay was the most reproducible of the three assays, with 1.2% error for SUVs and 3.9% error for LUVs. The dithionite assay also agreed best with theoretical inner:outer leaflet ratios, based on vesicle diameters determined by electron microscopy (Thomas et al. (1989) Biochem. Biophys. Acta 978, 85–90). Dithionite assay measurements were within 2.7% of theoretical ratios for SUVs and 2.3% for LUVs, while the NMR assay for SUVs was 14% lower than theoretical ratios and 23% lower for LUVs. The accuracy of NBD-lipids as markers for total transbilayer lipid was investigated. NBD-labeled phosphatidylserine, phosphatidylcholine and phosphatidylglycerol were accurate markers for total transbilayer lipid distribution, as their distributions were in close agreement with theoretical ratios. However, NBD-labeled phosphatidylethanolamine displayed a slight preference for the inner leaflet at low mole fractions of phosphatidylethanolamine, while native phosphatidylethanolamine showed a preference for the outer leaflet at the same concentration. NBD-labeled phosphatidic acid also showed a slight preference for the inner leaflet. We conclude that although dithionite-based assessment of NBD-labeled lipids across membrane bilayers can be a powerful analytical tool, caution must be used in the interpretation of results.     

Phospholipid vesicles promote human hemoglobin oxidation.

We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.     

CD Spectroscopy of Peptides and Proteins Bound to Large Unilamellar Vesicles

Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.     

Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Interaction of piroxicam and meloxicam with DMPG/DMPC mixed vesicles: anomalous partitioning behavior

Small unilamellar vesicles (SUVs) formed from a mixture of dimyristoylphosphatidylcholine (zwitterionic lipid with bulkier headgroup) and dimyristoylphosphatidylglycerol (anionic lipid with relatively smaller headgroup) allows better modulation of the physical properties of lipid bilayers compared to SUVs formed by a single type of lipid, providing us with a better model system to study the effect of membrane parameters on the partitioning of small molecules. Membrane parameter like packing of the vesicles is more pronounced in the gel phase and hence the study was carried out in the gel phase. Mixed vesicles formed from DMPG and DMPC with the mole percent ratio of 100:0, 90:10 and 80:20 were used for this study. As examples of polar solutes, piroxicam and meloxicam, two Non Steroidal Anti-inflammatory Drugs (NSAIDs) were chosen. The pH was adjusted to 2.8 in order to eliminate the presence of anionic forms of the drugs that would not approach the vesicles containing negatively charged DMPG (50% deprotonated at pH 2.8). Surface potential measured by using TNS (2,6-p-toluidinonaphthalene sulfonate, sodium salt) as surface charge sensitive probe showed no significant changes in the surface electrostatics in increasing DMPC content from 0 to 20%. Transmission electron microscopy (TEM) was used to characterize SUVs of different composition at pH 2.8. The average diameter of the mixed vesicles was found to be smaller than that formed by DMPG and DMPC alone. Partition coefficient (K(P)) of piroxicam and meloxicam was measured using intrinsic fluorescence of these molecules. K(P) value of piroxicam decreases with increase in DMPC content whereas it increases with DMPC content in case of meloxicam. This anomalous behavior of partitioning is unexpected since there was no significant change in surface pH of the vesicles and has been explained in terms of lipid packing and water penetration in the lipid bilayer.     

Mapping of the Lipid-Binding and Stability Properties of the Central Rod Domain of Human Dystrophin

Dystrophin is a cytoskeletal protein that confers resistance to the sarcolemma against the stress of contraction-relaxation cycles by interacting with cytoskeletal and membrane partners. Apart from several proteins, membrane phospholipids are a partner of the central rod domain made up of 24 spectrin-like repeats, separated into sub-domains by four hinges. We previously showed that repeats 1 to 3 bind to membrane anionic phospholipids, while repeats 20 to 24 are not able to do so. We focus here on the phospholipid-binding properties of the major part of the central rod domain, namely, the sub-domain delineated by hinges 2 and 3 comprising 16 repeats ranging from repeat 4 to 19 (R4-19). We designed and produced multirepeat proteins comprising three to five repeats and report their lipid-binding properties as well as their thermal stabilities. When these proteins are mixed with liposomes including the anionic lipid phosphatidylserine, they form stable protein-vesicle complexes as determined by gel-filtration chromatography. The absence of an anionic lipid precludes the formation of such complexes. Spectroscopic analyses by circular dichroism and tryptophan fluorescence show that, while the α-helical secondary structures are not modified by the binding, protein trans conformation leads to the movement of tryptophan residues into more hydrophobic environments. In addition, the decrease in the molar ellipticity ratio at 222/208 nm as observed by circular dichroism indicates that lipid binding reduces the inter-helical interactions of multirepeat proteins, thus suggesting partly “opened” coiled-coil structures. Combining these results with data from our previous studies, we propose a new model of the dystrophin molecule lying along the membrane bilayer, in which the two sub-domains R1-3 and R4-19 interact with lipids and F-actin, while the distal sub-domain R20-24 does not exhibit any interaction. These lipid-binding domains should thus maintain a structural link between cytoskeletal actin and sarcolemma via the membrane phospholipids.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Dissociation of peripheral protein-membrane complexes by high pressure.

The ability of high pressure to dissociate several peripheral protein-membrane complexes was investigated. Three vitamin K-dependent proteins (factor X, protein Z, and prothrombin) dissociated from small unilamellar vesicles (SUVs, 30 nm diameter) composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC) at comparable pressures (midpoints of 0.3-0.6 kbar). The pressure-induced dissociation curves for the factor X-SUV interaction followed the expected behavior for an interaction with an apparent dissociation equilibrium constant at atmospheric pressure, KD(atm), of 9 x 10(-7) M and a change in volume of association, delta Va, of 88 mL/mol. Factor X also dissociated from large unilamellar vesicles (LUVs, 100 nm diameter, 25% PS:75% PC) with a midpoint of 0.5 kbar. A second group of calcium-dependent membrane-binding proteins included protein kinase C (PKC), a 64-kDa protein, and a 32-kDa protein. The 32-kDa protein dissociated from SUVs (midpoint of 0.8 kbar), whereas PKC and the 64-kDa protein did not dissociate to a significant degree. The differences in dissociability of these proteins appeared to be a result of the differences in their KD(atm)'s (decreased dissociability with decreased KD(atm)). This pattern was further demonstrated by the relatively high midpoint of dissociation (1.1-1.4 kbar) of serum amyloid P component (SAP; KD(atm) ca. 10(-11)) and the limited dissociation of factor Va light chain (KD(atm) ca. 10(-11)). Changing the vesicle composition to phosphatidylethanolamine in place of PC gave higher affinity and decreased dissociation of the 32-kDa protein and SAP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of acid sphingomyelinase activity by lysosomal lipids and sphingolipid activator proteins

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substrate-carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent-free conditions only the sphingolipid activator protein SAP-C had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sphingomyelin degradation.     

Lipid peroxidation in small and large phospholipid unilamellar vesicles induced by water-soluble free radical sources

The susceptibility of small and large egg yolk phosphatidylcholine unilamellar vesicles to Fe(2+)/histidine-Fe(3+)- and Fenton reagent (Fe(2+)-H(2)O(2))-induced lipid peroxidation was evaluated by measuring the formation of thiobarbituric acid reactive substances (TBARS). It has been found that surface curvature or phospholipid packing exerts significant effect on the oxidative susceptibility of the unsaturated lipid bilayers and the highly curved and loosely packed small unilamellar vesicles (SUVs) exhibit much less resistance to the oxidative stress induced by the water-soluble free radical sources. The presence of lipid hydroperoxides in sonicated vesicles was excluded as the cause for higher level of lipid peroxidation in the phospholipid SUVs. Instead, the experimental results can be explained by the difference in ability of the water-soluble oxidants to penetrate the two types of lipid membranes. This hypothesis is supported by data obtained from fluorescence lifetime and quenching studies.     

Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Glycolipid transfer protein mediated transfer of glycosphingolipids between membranes: a model for action based on kinetic and thermodynamic analyses

Glycolipid transfer protein (GLTP) catalyzes the intermembrane transfer of lipids that have sugars beta-linked to either diacylglycerol or ceramide backbones, including simple glycosphingolipids (GSLs) and gangliosides. The present study provides a quantitative understanding of GLTP action involving bilayer vesicles that have high and low curvature stress, i.e., small and large unilamellar vesicles (SUVs and LUVs). When the GSL intervesicular transfer was monitored in real time using an established fluorescence resonance energy approach, the initial GSL transfer rates (v(0)) and net transfer equilibrium (K(eq)) were determined for GLTP-mediated transfer from SUVs and LUVs over the temperature range of 30-44 degrees C. v(0) exhibited a linear dependence with respect to varying GLTP concentrations (0-143 nM range) in SUVs and LUVs, suggesting a first order dependence on the GLTP bulk concentration. Thermodynamic parameters associated with the GLTP-GSL transition-state complex and GSL net transfer were determined from linear Arrhenius and van't Hoff plots, respectively. Although initial transfer rates were lower for LUVs than for SUVs, the activation energy barriers were higher for LUVs, while the Gibbs's free energy of the transition states were similar. The formation of a transition-state complex was predominantly enthalpy driven, whereas the net transfer of GSLs was mainly entropy driven. The rate-limiting step for GLTP action appeared to be the surface processes leading to the GLTP-GSL complex formation and release associated with a shuttle/carrier mode of action. Because surface processes leading to the GLTP-GSL complex formation were limiting for GLTP action with SUVs and LUVs, it was concluded that GLTP is likely to be a valuable tool to probe and manipulate GSL environments in membranes.