Physical determinants of β-barrel membrane protein folding in lipid vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)(60) and Opa(50) into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Electrodynamics of Lipid Membrane Interactions in the Presence of Zwitterionic Buffers

Due to thermal motion and molecular polarizability, electrical interactions in biological systems have a dynamic character. Zwitterions are dipolar molecules that typically are highly polarizable and exhibit both a positive and a negative charge depending on the pH of the solution. We use multilamellar structures of common lipids to identify and quantify the effects of zwitterionic buffers that go beyond the control of pH. We use the fact that the repeat spacing of multilamellar lipid bilayers is a sensitive and accurate indicator of the force balance between membranes. We show that common buffers can in fact charge up neutral membranes. However, this electrostatic effect is not immediately recognized because of the concomitant modification of dispersion (van der Waals) forces. We show that although surface charging can be weak, electrostatic forces are significant even at large distances because of reduced ionic screening and reduced van der Waals attraction. The zwitterionic interactions that we identify are expected to be relevant for interfacial biological processes involving lipid bilayers, and for a wide range of biomaterials, including amino acids, detergents, and pharmaceutical drugs. An appreciation of zwitterionic electrodynamic character can lead to a better understanding of molecular interactions in biological systems and in soft materials in general.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

Single-molecule imaging of the association of the cell-penetrating peptide Pep-1 to model membranes

Pep-1 is an amphiphatic peptide that can form noncovalent complexes with a cargo protein with subsequent delivery into a live cell. In this study, the behavior of Pep-1 was directly visualized by fluorescent imaging techniques at the single-molecule level of sensitivity. The interactions of Pep-1 and two of its labeled fluorescent analogues with large and cell-sized giant unilamellar vesicles and supported bilayers are reported. The role of the bilayer charge and ionic strength of the medium were examined. Pep-1 caused fusion and association of vesicles, and it perturbed the vesicle's membrane. The association of the peptide with neutral bilayers was promoted by anchoring of the cysteamine moiety. The association of the peptide with the structural defects of the neutral membrane was very efficient. The electrostatic forces were shown to be important for the association of the peptide only in low ionic strength solutions and were completely diminished at physiological ionic strength. Pep-1 did not induce the association to the model membrane of a number of proteins chosen to exhibit a range of properties. The results suggest that Pep-1 assisted delivery of cargo in living cells may result from cooperative effects.     

Spontaneous Curvature,Differential Stress,and Bending Modulus of Asymmetric Lipid Membranes

Lipid bilayers can exhibit asymmetric states, in which the physical characteristics of one leaflet differ from those of the other. This most visibly manifests in a different lipid composition, but it can also involve opposing lateral stresses in each leaflet that combine to an overall vanishing membrane tension. Here, we use theoretical modeling and coarse-grained simulation to explore the interplay between a compositional asymmetry and a nonvanishing differential stress. Minimizing the total elastic energy leads to a preferred spontaneous curvature that balances torques due to both bending moments and differential stress, with sometimes unexpected consequences. For instance, asymmetric flat bilayers, whose specific areas in each leaflet are matched to those of corresponding tensionless symmetric flat membranes, still exhibit a residual differential stress because the conditions of vanishing area strain and vanishing bending moment differ. We also measure the curvature rigidity of asymmetric bilayers and find that a sufficiently strong differential stress, but not compositional asymmetry alone, can increase the bending modulus. The likely cause is a stiffening of the compressed leaflet, which appears to be related to its gel transition but not identical with it. We finally show that the impact of cholesterol on differential stress depends on the relative strength of elastic and thermodynamic driving forces: if cholesterol solvates equally well in both leaflets, it will redistribute to cancel both leaflet tensions almost completely, but if its partitioning free energy prefers one leaflet over the other, the resulting distribution bias may even create differential stress. Because cells keep most of their lipid bilayers in an asymmetric nonequilibrium steady state, our findings suggest that biomembranes are elastically more complex than previously thought: besides a spontaneous curvature, they might also exhibit significant differential stress, which could strongly affect their curvature energetics.     

The structural organization of cationic lipid-DNA complexes

The interaction of cationic liposomes with supercoiled plasmid DNA results in a major rearrangement of each component to form compact multilamellar structures comprised of alternating layers of two-dimensional arrays of DNA sandwiched between lipid bilayers. Fluorescence resonance energy transfer was used to estimate the distance of closest approach of DNA to the lipid bilayers in these complexes. The effect of several compositional variables on this distance, including the ratio of cationic lipid to DNA, and the charge density, intrinsic curvature, and fluidity of the lipid bilayer were examined. Additionally, the effect of ionic strength was studied. For complexes prepared at or above a 3:1 charge ratio (+/-), the observed distance of closest approach was found to be in agreement with the intercalation of DNA between lipid bilayers. As the charge ratio was decreased, a monotonic increase in the distance was observed with a maximum observed at 0.5:1. Correlations between differences in the proximity of DNA to the lipid bilayer and the hydrodynamic size of the complexes were also found. A model based on these observations and previous reports suggests the formation of discrete populations of complexes below a charge ratio of 0.5:1 and above 3:1. The structure of the negatively charged complexes is consistent with DNA extending from the surface of the particles, whereas those possessing excess positive charge were multilamellar aggregates with the DNA effectively condensed between lipid bilayers. Complexes between these two states consist of weighted fractions of these two species.     

Molecular dynamics simulations of asymmetric NaCl and KCl solutions separated by phosphatidylcholine bilayers: potential drops and structural changes induced by strong Na+-lipid interactions and finite size effects

Differences of ionic concentrations across lipid bilayers are some of the primary energetic driving forces for cellular electrophysiology. While macroscopic models of asymmetric ionic solutions are well-developed, their connection to ion, water, and lipid interactions at the atomic scale are much more poorly understood. In this study, we used molecular dynamics to examine a system of two chambers of equal ionic strength, but differing amounts of NaCl and KCl, separated by a lipid bilayer. Our expectation was that the net electrostatic potential difference between the two chambers should be small or zero. Contrary to our expectation, a large potential difference (−70 mV) slowly evolved across the two water chambers over the course of our 172-ns simulation. This potential primarily originated from strong Na⁺ binding to the carbonyls of the phosphatidylcholine lipids. This ion adsorption also led to significant structural and mechanical changes in the lipid bilayer. We discuss this surprising result in the context of indirect experimental evidence for Na⁺ interaction with bilayers as well as potential caveats in current biomembrane simulation methodology, including force-field parameters and finite size effects.     

Role of hydrophobic forces in bilayer adhesion and fusion.

With the aim of gaining more insight into the forces and molecular mechanisms associated with bilayer adhesion and fusion, the surface forces apparatus (SFA) was used for measuring the forces and deformations of interacting supported lipid bilayers. Concerning adhesion, we find that the adhesion between two bilayers can be progressively increased by up to two orders of magnitude if they are stressed to expose more hydrophobic groups. Concerning fusion, we find that the most important force leading to direct fusion is the hydrophobic attraction acting between the (exposed) hydrophobic interiors of bilayers; however, the occurrence of fusion is not simply related to the strength of the attractive interbilayer forces but also to the internal bilayer stresses (intrabilayer forces). For all the bilayer systems studied, a single basic fusion mechanism was found in which the bilayers do not "overcome" their short-range repulsive steric-hydration forces. Instead, local bilayer deformations allow these repulsive forces to be "bypassed" via a mechanism that is like a first-order phase transition, with a sudden instability occurring at some critical surface separation. Some very slow relaxation processes were observed for fluid bilayers in adhesive contact, suggestive of constrained lipid diffusion within the contact zone.     

Cholesterol induced asymmetry in DOPC bilayers probed by AFM force spectroscopy

Cholesterol induced mechanical effects on artificial lipid bilayers are well known and have been thoroughly investigated by AFM force spectroscopy. However, dynamics of cholesterol impingement into bilayers at various cholesterol concentrations and their effects have not been clearly understood. In this paper we present, the effect of cholesterol as a function of its concentration in a simple single component dioleoylphosphatidylcholine (DOPC) bilayer. The nature of measured breakthrough forces on a bilayer with the addition of cholesterol, suggested that it is not just responsible to increase the mechanical stability but also introduces irregularities across the leaflets of the bilayer. This cholesterol induced asymmetry across the (in the inner and outer leaflets) bilayer is related to the phenomena of interleaflet coupling and is a function of cholesterol concentration probed by AFM can provide an unprecedented direction on mechanical properties of lipid membrane as it can be directly correlated to biophysical properties of a cell membrane.     

Lipid bilayer disruption by oligomeric α-synuclein depends on bilayer charge and accessibility of the hydrophobic core

Soluble oligomeric aggregates of α-synuclein have been implicated to play a central role in the pathogenesis of Parkinson's disease. Disruption and permeabilization of lipid bilayers by α-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer–membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged α-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer–membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer–membrane interactions.     

Electrophysiologic properties of channels induced by Abeta25-35 in planar lipid bilayers

Abeta25-35, a fragment of the neurotoxic amyloid beta protein Abeta1-42 found in the brain of Alzheimer patients, possesses amyloidogenic, neurotoxins and channel forming abilities similar to that of Abeta1-42. We have previously reported that Abeta25-35 formed voltage-dependent, relatively nonselective, ion-permeable channels in planar lipid bilayers. Here, we show that Abeta25-35 formed channels in both solvent-containing and solvent-free bilayers. We also report that for Abeta25-35, channel forming activity was dependent on ionic strength, membrane lipid composition, and peptide concentration, but not on pH. Lower ionic strength and negatively charged lipids increased channel formation activity, while cholesterol decreased activity. The nonlinear function relating [Abeta25-35] and membrane activity suggests that aggregation of at least three monomers is required for channel formation.     

Cytochrome c adsorption to supported, anionic lipid bilayers studied via atomic force microscopy

The adsorption of membrane-associated protein cytochrome c to anionic lipid bilayers of dioleoyl phosphatidylglycerol was studied in low ionic strength physiological buffer using atomic force microscopy. The bilayers were supported on polylysinated mica. The formation of stable, single lipid bilayers was confirmed by imaging and force spectroscopy. Upon addition of low concentrations of cytochrome c, protein molecules were not topographically visible on the lipid bilayer-buffer interface. However, the forces required to punch through the bilayer by indentation using the atomic force microscopy probe were significantly lower after protein adsorption, which suggest that the protein inserts into the bilayer. Moreover, the apparent thickness of the bilayer remained unchanged after cytochrome c adsorption. Yet, mass spectroscopy and visible light absorption spectroscopy confirmed the presence of cytochrome c in the lipid bilayers. These results suggest that 1), cytochrome c inserts into the bilayer and resides in its hydrophobic core; 2), cytochrome c insertion changes the mechanical properties of the bilayer significantly; and 3), bilayer force spectroscopy may be a useful tool in investigating lipid-protein interactions.     

Effect of Physical Parameters on the Main Phase Transition of Supported Lipid Bilayers

Supported lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) were assembled by the vesicle fusion technique on mica and studied by temperature-controlled atomic force microscopy. The role of different physical parameters on the main phase transition was elucidated. Both mixed (POPE/POPG 3:1) and pure POPE bilayers were studied. By increasing the ionic strength of the solution and the incubation temperature, a shift from a decoupled phase transition of the two leaflets, to a coupled transition, with domains in register, was obtained. The observed behavior points to a modulation of the substrate/bilayer and interleaflet coupling induced by the environment and preparation conditions of supported lipid bilayers. The results are discussed in view of the role of different interactions in the system. The influence of the substrate on the lipid bilayers, in terms of interleaflet coupling, can also help us in understanding the possible effect that submembrane elements like the cytoskeleton might have on the structure and dynamics of biomembranes.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Insertion and pore formation driven by adsorption of proteins onto lipid bilayer membrane-water interfaces

We describe the binding of proteins to lipid bilayers in the case for which binding can occur either by adsorption to the lipid bilayer membrane-water interface or by direct insertion into the bilayer itself. We examine in particular the case when the insertion and pore formation are driven by the adsorption process using scaled particle theory. The adsorbed proteins form a two-dimensional "surface gas" at the lipid bilayer membrane-water interface that exerts a lateral pressure on the lipid bilayer membrane. Under conditions of strong intrinsic binding and a high degree of interfacial converge, this pressure can become high enough to overcome the energy barrier for protein insertion. Under these conditions, a subtle equilibrium exists between the adsorbed and inserted proteins. We propose that this provides a control mechanism for reversible insertion and pore formation of proteins such as melittin and magainin. Next, we discuss experimental data for the binding isotherms of cytochrome c to charged lipid membranes in the light of our theory and predict that cytochrome c inserts into charged lipid bilayers at low ionic strength. This prediction is supported by titration calorimetry results that are reported here. We were furthermore able to describe the observed binding isotherms of the pore-forming peptides endotoxin (alpha 5-helix) and of pardaxin to zwitterionic vesicles from our theory by assuming adsorption/insertion equilibrium.     

The Basic Protein of CNS Myelin: Its Structure and Ligand Binding

Consideration of the evidence presented in this review leads to the following conclusions: (a) Isolated MBP in aqueous solution has little ordered secondary or tertiary structure. (b) In this state, the protein can associate with a wide range of hydrophobic and amphiphilic compounds, these interactions involving limited sections of the protein. (c) The strength of binding to bilayers and the accompanying conformational changes in the protein are greatest for systems containing acidic lipids, presumably because of the involvement of ionic interactions. (d) When bound to bilayers of acidic lipids, MBP will have substantially more ordered secondary structure than it manifests in aqueous solution, and it is likely to be oligomeric (possibly hexameric). (e) MBP does affect the organization of lipid aggregates. It influences strongly the separation of bilayers in multilayers of purified lipids, and at present this must be viewed as its prime role within myelin. The greatest impediment to our understanding of MBP is the lack of an assayable biological activity. In contrast to the situation with enzymes, for example, we have no functional test for changes in protein structure or changes accompanying interactions with other molecules. Current evidence suggests that the protein has a structural role within myelin and that its own three-dimensional structure is strongly dependent on the molecules with which it is associated. If this picture is correct, studies of the isolated protein or of the protein in reconstituted lipid systems may yield, at best, a rough guide to the structure within its biological environment. Further clarification of the structure and function of MBP may have to await development of more powerful techniques for studying proteins bound to large molecular aggregates, such as lipid bilayers. The paucity of generally applicable methods is reflected in the fact that even low resolution structures are known for only a handful of intrinsic membrane proteins, and even more limited information exists for proteins associated with membrane surfaces. However, the increasing use of a combination of electron microscopy and diffraction on two-dimensional arrays of proteins formed on lipid bilayers (Henderson et al., 1990) offers the hope that it may not be too long before it will be possible to study at moderate resolution the three-dimensional structure of MBP bound to a lipid membrane.     

Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.     

pH-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly alpha-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel beta-sheet structures at the membrane surface. The two forms are readily interconvertible and the equilibrium between them is determined by the pH and ionic strength of the surrounding solution. Lowering the pH favors the monomeric alpha-helical conformation, whereas increasing the ionic strength shifts the equilibrium towards the membrane-associated beta-aggregates. The binding data are interpreted in terms of a cooperative binding model that yields free energies of insertion and free energies of self-association for each of the peptides studied at pH 7.4 and pH 5. At pH 5 and 35 mM ionic strength, the insertion energy of the 20 residue influenza hemagglutinin fusion peptide is -7.2 kcal/mol and the self-association energy is -1.9 kcal/mol. We propose that self-association of fusion peptides could be a major driving force for recruiting a small number of hemagglutinin trimers into a fusion site.