Negative Spontaneous Curvature Research Articles

Interactions of a bacterial trehalose lipid with phosphatidylglycerol membranes at low ionic strength

Trehalose lipids are bacterial biosurfactants which present interesting physicochemical and biological properties. These glycolipids have a number of different commercial applications and there is an increasing interest in their use as therapeutic agents. The amphiphilic nature of trehalose lipids points to the membrane as their hypothetical site of action and therefore the study of the interaction between these biosurfactants and biological membranes is critical. In this study, we examine the interactions between a trehalose lipid (TL) from Rhodococcus sp. and dimyristoylphosphatidylglycerol (DMPG) membranes at low ionic strength, by means of differential scanning calorimetry, light scattering, fluorescence polarization and infrared spectroscopy. We describe that there are extensive interactions between TL and DMPG involving the perturbation of the thermotropic intermediate phase of the phospholipid, the destabilization and shifting of the DMPG gel to liquid crystalline phase transition to lower temperatures, the perturbation of the sample transparency, and the modification of the order of the phospholipid palisade in the gel phase. We also report an increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer. These changes would increase the monolayer negative spontaneous curvature of the phospholipid explaining the destabilizing effect on the intermediate state exerted by this biosurfactant. The observations contribute to get insight into the biological mechanism of action of the biosurfactant and help to understand the properties of the intermediate phase display by DMPG at low ionic strength.

Effective interaction between small unilamellar vesicles as probed by coarse-grained molecular dynamics simulations

Abstract A series of molecular dynamics (MD) simulations has been undertaken to investigate the effective interaction between vesicles including PC (phosphatidylcholine) and PE (phosphatidylethanolamine) lipids using the Shinoda–DeVane–Klein coarse-grained force field. No signatures of fusion were detected during MD simulations employing two apposed unilamellar vesicles, each composed of 1512 lipid molecules. Association free energy of the two stable vesicles depends on the lipid composition. The two PC vesicles exhibit a purely repulsive interaction with each other, whereas two PE vesicles show a free energy gain at the contact. A mixed PC/PE (1:1) vesicle shows a higher flexibility having a lower energy barrier on the deformation, which is caused by lipid sorting within each leaflet of the membranes. With a preformed channel or stalk between proximal membranes, PE molecules contribute to stabilize the stalk. The results suggest that the lipid components forming the membrane with a negative spontaneous curvature contribute to stabilize the stalk between two vesicles in contact.

The Effect of the Hydrophobic Surfactant Proteins on HII-Curvature Depends on the Cylindrical Radius

Several small hydrophobic peptides can promote the fusion of lipid bilayers. The stalk model proposes that fusion proceeds via a negatively curved intermediate, and suggests that the peptides might achieve their fusogenic effect by enhancing negative spontaneous curvature of the individual lipid leaflets. Direct measurements on the unpaired monolayers of the inverse hexagonal (HII) phase have shown that the peptides can induce more negative curvature, but to a limited extent. The hydrophobic surfactant proteins, SP-B and SP-C, show similar behavior. They promote the adsorption of vesicles to an air/water interface by a process analogous to fusion. They enhance the negative spontaneous curvature of HII monolayers formed by dioleoyl phosphatidylethanolamine (DOPE):dioleoyl phosphatidylglycerol (9:1, mol:mol), but only by 8%. The studies here tested whether the limited response might reflect the size of the HII cylinder and the restricted space available for incorporation of the proteins. Small angle x-ray scattering showed that different ratios of DOPE with dioleoyl phosphatidylcholine (DOPC) produced HII cylinders with different radii. The effects of the proteins depended on the cylindrical size. Up to 3% (w:w) of the proteins produced no change in the smallest cylinders, formed by DOPE alone. With larger cylinders, the proteins reduced the radius. The magnitude of the change increased with cylindrical size. At 80% (mol:mol) DOPC, 5% (wt:wt) protein reduced the outer cylindrical radius by 78%. This change represented a 90% increase in the estimated magnitude of negative spontaneous curvature. These results show that demonstrating the ability of the hydrophobic surfactant proteins to promote negative curvature requires HII cylinders above a minimum size. Our findings raise the possibility that the effects on HII-curvature of other fusogenic peptides might also depend on the cylindrical radius. Acknowledgements: NIH; SSRL.

Membrane shape changes at initial stage of membrane fusion under the action of proteins inducing spontaneous curvature

This paper studies change of membrane shape at the initial stage of the fusion process due to the fusion proteins inducing spontaneous curvature in the membrane. As protein inclusions are embedded into the membrane, a highly curved surface forms in the center of the membrane; it facilitates the formation of short-lived hydrophobic defects and leads to the merger of the contact monolayers of the membranes. Membrane is considered as continuous liquid-crystal medium subject to elastic deformations. One deformational mode of splay is taken into account; energy is calculated in the quadratic approximation on this deformation. In case of positive spontaneous curvature induced by the protein there is no bulge on the top of the membrane despite high deviation of membrane shape from the equilibrium state. In case of negative spontaneous curvature a bulge is formed and its height and curvature increase with the increase of the membrane curvature in the initial state.

Cytochrome c causes pore formation in cardiolipin-containing membranes

The release of cytochrome c from mitochondria is a key signaling mechanism in apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanisms remain unclear. Cytochrome c (cyt c) binds to and penetrates lipid structures containing the inner mitochondrial membrane lipid cardiolipin (CL), leading to protein conformational changes and increased peroxidase activity. We describe here a direct visualization of a fluorescent cyt c crossing synthetic, CL-containing membranes in the absence of other proteins. We observed strong binding of cyt c to CL in phospholipid vesicles and bursts of cyt c leakage across the membrane. Passive fluorescent markers such as carboxyfluorescein and a 10-kDa dextran polymer crossed the membrane simultaneously with cyt c, although larger dextrans did not. The data show that these bursts result from the opening of lipid pores formed by the cyt c-CL conjugate. Pore formation and cyt c leakage were significantly reduced in the presence of ATP. We suggest a model, consistent with these findings, in which the formation of toroidal lipid pores is driven by initial cyt c-induced negative spontaneous membrane curvature and subsequent protein unfolding interactions. Our results suggest that the CL-cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis.

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in Membranes Depends on the Lipid Spontaneous Curvature

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state 15N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.

IFITM Proteins Restrict Viral Membrane Hemifusion

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

An Anionic Phospholipid Enables the Hydrophobic Surfactant Proteins to Alter Spontaneous Curvature

The cationic, hydrophobic surfactant proteins, SP-B and SP-C (SPs), promote adsorption of vesicles to an air water interface and greatly increase the rate of forming an alveolar film. The available evidence suggests that the SPs may facilitate adsorption by promoting the generation of a negatively curved intermediate. The studies here tested whether the proteins could change the curvature of phospholipid leaflets in the inverse hexagonal (HII) phase. Experiments with the uncharged dioleoyl phosphatidylethanolamine (DOPE) showed no change in the lattice-constant with added SPs. Pulmonary surfactant contains ∼10% (mol:mol) anionic phospholipids. We therefore also examined mixtures of DOPE with 10% (mol:mol) of the anionic dioleoyl phosphatidylglycerol (DOPG) to investigate selective interactions with the SPs. Small angle X-ray scattering established the phase diagram (temperature - % protein) and lattice-constants for these lipids combined with the physiological mixture of the SPs. The hexagonal lattice-constant decreased linearly with increasing amounts of protein before reaching a temperature-dependent maximum change of 8-14% at ∼1% (w:w) protein. To test whether this effect was strictly electrostatic, NaCl concentrations up to 3M provided electrostatic screening. The salt diminished but did not eliminate the dose-related change in the lattice-constant. Measurements at different hydrations showed that the separation between the pivotal plane and the aqueous core was unaffected by the proteins, indicating that the change in the lattice-constant produced by the proteins reflects a more negative spontaneous curvature. These results provide direct evidence that the hydrophobic surfactant proteins can enhance the negative curvature of lipid leaflets. Studies were conducted at the Stanford Synchrotron Radiation Lightsource.

IFITM Proteins Block the Creation of Hemifusion

Interferon-inducible transmembrane (IFITM) proteins have previously been shown to potently reduce infection caused by several highly pathogenic viruses, but the mechanisms of inhibition were not identified. There are three known functional IFITM members that are widely expressed in human tissues and cell lines. The extent of inhibition of infectivity varies among the functional IFITM-members. We have found that the presence of IFITM proteins blocks viral-protein-induced fusion in a pattern that is dependent on the cell type expressing the fusion protein. IFITM1 blocks fusion and IFITM2 does not, independent of cell line. The IFITM3 block does depend on the cell line expressing the fusion protein. The pattern of blockage was the same for viral fusion proteins of the three structural classes of fusion proteins, indicating that the block occurs through a physical mechanism, rather than being dependent on specific structural or chemical properties. We performed cell-cell fusion experiments for low-pH-requiring proteins to determine the intermediate point at which inhibition occurs. We acidified at low temperature to create a state of hemifusion and found that subsequent raising of temperature or addition of chlorpromazine (CPZ) did not cause fusion for IFITM1 target cells. We conclude that the interferon-inducible protein blocks fusion at a step prior to hemifusion. Experiments were performed in which oleic acid (OA) was added prior to or subsequent to creating the hemifusion intermediate to determine whether the negative spontaneous curvature conferred by OA could relieve the block caused by IFITM. OA promoted fusion only if present prior to creating the hemifusion intermediate, showing that this amphiphile facilitated the occurrence of hemifusion. We conclude that the physical state of the lipid bilayer is altered by IFITM proteins, and this change underlies the inhibition of fusion and infectivity.

Effects of Flavonols on the Magnitude of Dipole Potential of Phospholipid Bilayers

The changes in K+-nonactin steady-state membrane conductance were measured to estimate the changes in the dipole potential (Δφd) of phospholipid bilayers (DOPS, DOPE, DOPC) after two-side addition of quercetin or myricetin into membrane bathing solution (0.1M KCl pH7.4). Table presents the results of the approximation of the obtained data by Langmuir adsorption isotherm. The maximum dipole potential change Δφd(∞) depends on flavonol and membrane types. It is larger in case of quercetin than for myricetin. This difference may be attributed to higher hydrophobicity of quercetin compared with myricetin. The smaller values of |Δφd(∞)| in the case of DOPS and DOPE than for DOPC may be resulted from smaller effective surface density of dipoles due to electrostatic repulsion between serine residues and negative spontaneous curvature of DOPE relative to membranes made from the phospholipid with neutral spontaneous curvature, DOPC. Comparison of dissociation constants (K) shows that flavonol affinity for DOPE-bilayers is smaller than that for DOPS and DOPC-membranes. The study was supported in part by RFBR, the Grant of the President of RF (#MK-1813.2012.4) and the Russian State Contract #8119 (MES, FTP, SSEPIR).

Synergistic Insertion of Antimicrobial Peptides into Membranes

The antimicrobial peptides (AMPs) PGLa and magainin 2 (MAG2) found in the skin of the African frog Xenopus laevis show a synergistic enhancement of their activity [1]. Both peptides form amphipathic α-helices when binding to a lipid membrane, and their orientation in membranes have been determined with high accuracy using solid state 2H-, 15N-, and 19F-NMR.We have previously shown that PGLa inserts into DMPC/DMPG bilayers in the presence of an equimolar amount of MAG2, but not on its own, even at high concentrations [2]. This indicates formation of stable heterodimeric peptide pores, which in turn can explain the synergism between the peptides. Here, we show that MAG2, both with and without PGLa, always stays almost flat on the membrane surface; however, there is a small change in the orientation in the presence of PGLa, and dynamics is reduced, indicating that a PGLa-MAG2 complex is formed. In POPC/POPG, both peptides stay flat on the membrane surface, alone or combined. This is in agreement with our previous finding that the AMP MSI-103 always stays flat on the surface in unsaturated lipids (or more generally in lipid systems with negative spontaneous curvature), but can insert deeper into the membrane in saturated lipids (where the spontaneous curvature is positive) [3].

Rhodopsin Activation is Modulated by Non-Specific Membrane Lipid-Protein Interactions

Rhodopsin activation proceeds through an ensemble of conformational substates: Meta I ↔ Meta IIa ↔ Meta IIb ↔ Meta IIbH+ [1]. These substates are characterized by Schiff base deprotonation (Meta I), an outward tilt of helix H6 (Meta IIb), and protonation of Glu134 (Meta IIbH+). Lipid bilayer composition has been shown to affect rhodopsin activation, providing an opportunity to systematically investigate membrane protein-lipid bilayer interactions on the mesoscale [2]. To quantify lipid acyl chain effects on GPCR activation, rhodopsin was reconstituted in lipids with symmetric unsaturated acyl chains (DOPC) or asymmetric lipids with sn-1 saturated and sn-2 unsaturated acyl groups (POPC). Proteolipid recombinant membranes were studied by UV-visible and FTIR spectroscopy. Symmetric membrane lipids (DOPC) stabilize Meta IIa and render the normally weakly populated substate accessible to study. The Meta IIa substate is characterized by an opening of the Schiff base ionic lock, and an activation switch in a conserved water-mediated, hydrogen-bonded network involving helices H1/H2/H7 that is sensed by Asp83. Replacement of an unsaturated acyl chain with a saturated chain (POPC) increases the pKa value for the Meta I ↔ Meta II equilibrium, and consequently destabilizes the Meta IIa substate. Modulation of the bilayer curvature stress due to a negative monolayer spontaneous curvature (H0) (DOPC) contributes a mechanical force that shifts the Meta I ↔ Meta II equilibrium towards Meta II, leading to rhodopsin activation. The flexible surface model (FSM) explains how chemically non-specific interactions between membrane proteins and the lipid bilayer contribute to GPCR activation [3,4].[1] M. Mahalingam et al. (2008) PNAS 105:17795.[2] E. Zaitseva et al. (2010) JACS 132:4815.[3] A.V. Botelho et al. (2006) Biophys. J. 91:4464.[4] M.F. Brown (1994) Chem. Phys. Lipids 73:159.

Time-Resolved UV-Visible Studies of Rhodopsin Provide Experimental Test of Flexible Surface Model for Lipid-Protein Interactions

Time-resolved UV/Visible absorbance of the retinal chromophore of rhodopsin [1] delivers vital information about key GPCR activation steps in a membrane lipid environment [2]. Following rhodopsin photoexcitation, a minimum of four intermediates equilibrate on the millisecond-time scale [2,3]. The first equilibrium is between the protonated Schiff base (PSB) species Meta I480 and the deprotonated SB species Meta IIa and is pH independent. A second equilibrium entails spectrally silent conversion of Meta IIa to the Meta IIb substate. The final step is protonation of Glu134 of the ERY sequence in Meta IIb to yield Meta IIbH+ whose pKa characterizes the acid-base equilibrium [3]. Absorbance measurements on the microsecond-to-hundred millisecond-time scale allowed us to study effects of POPC, DOPC, or DOPC/DOPE mixtures on the first equilibrium constant K1 and on the pKa of the final equilibrium. Results were analyzed by singular-value decomposition and were fit by a linear combination of temperature-dependent basis spectra. Notably an increase in K1 was discovered due to either PE head groups or increased acyl chain unsaturation, whereas little change in pKawas evident. According to the flexible-surface model (FSM) rhodopsin becomes a sensor of negative spontaneous (intrinsic) curvature upon light activation [4]. Competition between the curvature elastic energy and the hydrophobic mismatch at the proteolipid boundary explains the influences of lipid-protein interactions [4]. Both the lipid acyl chains and polar head groups affect the Meta I-Meta II transition, revealing how protein energetics are affected by material properties of the lipid bilayer.[1] A.V. Struts et al. (2011) Nature Struct. Mol. Biol. 18, 392-394.[2] J. Epps et al. (2006) Photochem. Photobiol. 82, 1436-1441.[3] M. Mahalingam et al. (2008) PNAS 105 17795-17800.[4] A. V. Botelho et al. (2006) Biophys. J. 91, 4464-4477.

Flexible Surface Model for Membrane Lipid-Protein Interactions

Membrane protein conformational changes, folding and stability, and membrane fusion may all involve elastic deformation of the bilayer. The development of solid-state deuterium NMR spectroscopy [1] provides a basis for experimentally investigating lipid structure and dynamics under external forces due to temperature, osmotic pressure, or lipid composition. Deuterium NMR relaxation illuminates long-range collective lipid interactions [1]. Non-specific properties of the bilayer play a significant role in modulating protein conformational energetics [2]. A flexible-surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. The FSM describes elastic coupling of membrane lipids to integral membrane proteins. Curvature and hydrophobic matching to the lipid bilayer entails a stress field that explains membrane protein activity and stability [3]. Rhodopsin provides an important example, where solid-state NMR studies [4,5] and FTIR spectroscopy [6] characterize the energy landscape of the dynamically activated receptor. Upon light activation rhodopsin becomes a sensor of negative spontaneous curvature [2]. Time-resolved UV-visible and FTIR spectroscopic studies show how membrane lipids forward or back-shift the metarhodopsin equilibrium due to chemically non-specific material properties [2]. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on rhodopsin function are all explained by the new biomembrane model. By contrast, the older fluid-mosaic model including lipid rafts fails to account for such effects on membrane protein activity. According to the FSM, proteins are regulated by membrane lipids whose spontaneous curvature most closely matches the activated state within the lipid membrane. [1] M.F. Brown (1996) in Membrane Structure & Dynamics, pp. 175-252. [2] M.F. Brown (1994) Chem. Phys. Lipids 73, 159-180. [3] A.V. Botelho et al. (2006) Biophys. J. 91, 4464. [4] A.V. Struts et al. (2011) Nature Struct. Mol. Biol. 18, 392-394. [5] A.V. Struts et al. (2011) PNAS 20, 8263-8268. [6] M. Mahalingam et al. (2008) PNAS 105, 17795-17800.

Simulation of fusion-mediated nanoemulsion interactions with model lipid bilayers

Perfluorocarbon-based nanoemulsion particles have become promising platforms for the delivery of therapeutic and diagnostic agents to specific target cells in a non-invasive manner. A "contact-facilitated" delivery mechanism has been proposed wherein the emulsifying phospholipid monolayer on the nanoemulsion surface contacts and forms a lipid complex with the outer monolayer of target cell plasma membrane, allowing cargo to diffuse to the surface of target cell. While this mechanism is supported by experimental evidence, its molecular details are unknown. The present study develops a coarse-grained model of nanoemulsion particles that are compatible with the MARTINI force field. Simulations using this coarse-grained model have demonstrated multiple fusion events between the particles and a model vesicular lipid bilayer. The fusion proceeds in the following sequence: dehydration at the interface, close apposition of the particles, protrusion of hydrophobic molecules to the particle surface, transient lipid complex formation, absorption of nanoemulsion into the liposome. The initial monolayer disruption acts as a rate-limiting step and is strongly influenced by particle size as well as by the presence of phospholipids supporting negative spontaneous curvature. The core-forming perfluorocarbons play critical roles in initiating the fusion process by facilitating protrusion of hydrophobic moieties into the interface between the two particles. This study directly supports the hypothesized nanoemulsion delivery mechanism and provides the underlying molecular details that enable engineering of nanoemulsions for a variety of medical applications.

Membrane solubilisation and reconstitution by octylglucoside: Comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry

We have studied the solubilisation and reconstitution of lipid membranes composed of either synthetic phosphatidylcholine or Escherichia. coli polar lipid extract by the non-ionic detergent octylglucoside. For both lipid systems, composition-dependent transformations of unilamellar vesicles into micelles or vice versa were followed by high-sensitivity isothermal titration calorimetry. Data obtained over a range of detergent and lipid concentrations could be rationalised in terms of a three-stage phase separation model involving bilayer, bilayer/micelle coexistence, and micellar ranges, yielding the detergent/lipid phase diagrams and the bilayer-to-micelle partition coefficients of both detergent and lipid. The most notable difference between the lipids investigated was a substantial widening of the bilayer/micelle coexistence range for E. coli lipid, which was due to an increased preference of the detergent and a decreased affinity of the lipid for the micellar phase as compared with the bilayer phase. These effects on the bilayer-to-micelle partition coefficients could be explained by the high proportion in E. coli membranes of lipids possessing negative spontaneous curvature, which hampers both their transfer into strongly curved micellar structures as well as the insertion of detergent into condensed bilayers.

The Intermediate State of DMPG Is Stabilized by Enhanced Positive Spontaneous Curvature

1,2-Dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG) at low salt concentrations has a complex endotherm with at least four components and extending over the span of 20 degrees. During this ongoing melting, the solution becomes viscous and scatters light poorly. This multipeak endotherm was suggested to result from the effects of curvature on the relative free energies of gel and fluid DMPG bilayers, further relating to the formation of an intermediate sponge phase between the lamellar gel and fluid phases. Although later studies appear to exclude a connected bilayer network, the relation of the endotherm peaks to curvature remains an appealing hypothesis. This was tested by including in the system both water-soluble small molecules (dimethyl sulfoxide, ethanol, and urea) as well as amphiphiles (myristoyl-lyso-PG, cholesterol, cholesterol-3-sulfate, and dimyristoylglycerol) known to alter the spontaneous curvature of bilayers. All compounds increasing the monolayer positive spontaneous curvature (ethanol, urea, myristoyl-lyso-PG, cholesterol-3-sulfate) increased the temperature span of the intermediate state and elevated the temperature of its dissolution, while all compounds increasing the negative spontaneous curvature (dimethyl sulfoxide, cholesterol, dimyristoylglycerol) had the opposite effect, implying that the intermediate state contains a structure with positive curvature. The results support the view that the intermediate state consists of vesicles with a large number of holes. The viscosity increase could be related to vesicle expansion needed to accommodate the numerous holes.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Deformation of Vesicles Controlled by Local Spontaneous Curvatures of Membrane

The metabolic pathways are fundamental processes to maintain the life, which is supported by the cell membrane deformations such as, membrane adhesion, fusion and pore formation. In the present living organisms, the membrane deformations in the metabolic pathways are conducted by the related proteins. However, we believe that such membrane deformations can be caused by controlling the physical properties of the membrane without the aid of proteins. In this context we demonstrate the deformations of vesicle by controlling the spontaneous curvature of lipids.First, we focused on the lipids with negative spontaneous curvature (NL: DPPE and DPhPC). The binary vesicles composed of NL and lipids with zero spontaneous curvature (ZL: DOPC and DPPC) show a phase separation between NL rich domains and ZL rich matrix under the immiscibility temperature. When the two phase separating vesicles are contacted using micropipettes, they adhere each other through the domain rich in NL.Second, we have investigated the effects of the lipids with positive spontaneous curvature (PL: DHPC) on the shape of vesicles. The binary vesicles composed of PL and ZL form spherical shape in one-phase region. By decreasing the temperature, the binary vesicles show a burst and then form a single pore on the vesicle. There are three types of pores, simple circular, rolled-rim and wrinkle-rim, depending on the ratio of PL to ZL. The pore closes with increase the temperature and finally vesicles return to the spherical shape again. We discuss this shape deformation of vesicles by calculating time development equation of the membrane free energy.We believe that the control of the spontaneous curvature of lipids is a key to realize the model metabolic pathways without proteins.

Modulation of Membrane Rigidity by Sar1, a Vesicle Trafficking Protein

Intracellular cargo trafficking involves dramatic changes in membrane shape, the mechanics of which remain poorly understood. We focus on Sar1, the key regulator of the coat protein complex II (COPII) family that ferries newly synthesized proteins from the endoplasmic reticulum (ER), and the only member of the COPII coat that interacts directly with the ER lipid bilayer membrane. To investigate whether Sar1 has a role beyond merely localizing the other COPII proteins, we directly measure the force involved in membrane deformation as a function of its concentration, using optically trapped microspheres to pull tethers from in vitro lipid membranes whose composition and large surface area mimic the composition and geometry of the ER. We find that Sar1 lowers the rigidity (bending modulus) of lipid membranes to nearly zero in a concentration-dependent manner. Moreover, Sar1 lacking its N-terminal amphipathic helix induces negative (concave) spontaneous membrane curvature. These results reveal a paradigm-altering insight into COPII trafficking: Sar1 actively alters the material properties of the membranes it binds to, lowering the energetic cost of curvature generation.View Large Image | View Hi-Res Image | Download PowerPoint Slide