Physical Properties of Escherichia coli Spheroplast Membranes

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.     

Mechanosensitivity of gramicidin A channels in semispherical bilayer membranes at constant tension

Mechanoelectrical transduction in gramicidin A channels was studied in macroscopic planar lipid bilayer membranes bulged at constant tension. We found a supralinear increase in the single channel activity, which was proportional to the square of membrane radius but could not be accounted for by the increase in membrane surface area or by recruitment of new channels. When being extrapolated to biological membranes, these observations may suggest that the activity of permeability of ion channels can be influenced simply by changing the shape of the membrane, with or without stretching.     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.     

Mechanosensitivity of gramicidin A channels in bulged bilayer membranes at constant tension

Mechanoelectrical transduction in gramicidin A channels was studied in macroscopic planar lipid bilayer membranes bulged at constant tension. We found a supralinear increase in the single channel activity that was proportional to the square of membrane radius, but could not be accounted for by the increase in membrane surface area, or by recruitment of new channels. Extrapolated to biological membranes, these observations may suggest that the permeability of ion channels can be influenced simply by changing shape of the membrane, with or without stretching. Published in Russian in Biofizika, 2006, Vol. 51, No. 6, pp. 1014–1018. The text was submitted by the authors in English.     

Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm² in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.     

Dynamic Response of Model Lipid Membranes to Ultrasonic Radiation Force

Low-intensity ultrasound can modulate action potential firing in neurons in vitro and in vivo. It has been suggested that this effect is mediated by mechanical interactions of ultrasound with neural cell membranes. We investigated whether these proposed interactions could be reproduced for further study in a synthetic lipid bilayer system. We measured the response of protein-free model membranes to low-intensity ultrasound using electrophysiology and laser Doppler vibrometry. We find that ultrasonic radiation force causes oscillation and displacement of lipid membranes, resulting in small (<1%) changes in membrane area and capacitance. Under voltage-clamp, the changes in capacitance manifest as capacitive currents with an exponentially decaying sinusoidal time course. The membrane oscillation can be modeled as a fluid dynamic response to a step change in pressure caused by ultrasonic radiation force, which disrupts the balance of forces between bilayer tension and hydrostatic pressure. We also investigated the origin of the radiation force acting on the bilayer. Part of the radiation force results from the reflection of the ultrasound from the solution/air interface above the bilayer (an effect that is specific to our experimental configuration) but part appears to reflect a direct interaction of ultrasound with the bilayer, related to either acoustic streaming or scattering of sound by the bilayer. Based on these results, we conclude that synthetic lipid bilayers can be used to study the effects of ultrasound on cell membranes and membrane proteins.     

How cholesterol is distributed between monolayers in asymmetric lipid membranes

The distribution of cholesterol in asymmetric lipid bilayers was studied by extensive coarse-grained molecular dynamics simulations. The effects of the lipid head group charge, acyl chain saturation, spontaneous membrane curvature and surface tension of the membrane were investigated. Four asymmetric bilayers containing DOPC, DOPS, DSPC or DSPS lipids were simulated on a time scale extended to tens of microseconds. We show that cholesterol strongly prefers anionic lipids to neutral and saturated lipid tails to unsaturated with a distribution ratio of ~0.7 in neutral/anionic bilayers and of ~0.4 in unsaturated/saturated bilayers. Multiple flip-flop transitions of cholesterol were observed directly, and their mean times ranged from 80 to 250?ns. It was shown that the distribution of cholesterol in the asymmetric membrane depends not only on the type of lipid, but also on the local membrane curvature and the surface tension. The membrane curvature enhances the influence of the lipid head groups on cholesterol distribution, while non-optimal surface tension caused by different areas per lipid in different monolayers increases the effect of the lipid tail saturation. It was clearly seen that the monolayers of asymmetric bilayers are interdependent. Mean distances from the bilayer center to cholesterol molecules depend not only on the type of the lipid in the considered monolayer but also on the composition of the opposite monolayer.     

Conformational and orientation studies of artificial ion channels incorporated into lipid bilayers

The conformational and orientation studies in lipid bilayers of 21 amino acid peptides bearing six crown ethers are reported. The compounds were designed to form artificial ion channels by stacking the crown rings, and were shown to be functional in bilayer membranes. We used Fourier transform infrared spectroscopy and CD spectropolarimetry to study the conformation of the peptides in solution and in lipid bilayers. These studies revealed that hexacrown peptides retain their alpha-helical conformation when incorporated in a lipid bilayer environment. Attenuated total reflectance spectroscopy was used to investigate the orientation of the peptides in a lipid bilayer. Results demonstrated that the peptides are not oriented at a fixed angle in membrane, but rather are in incorporation equilibrium between an active state parallel to the lipid chain and an inactive state adsorbed at the surface of the bilayer. From these results, we propose a model for the channel activity and the gating mechanism of these hexacrown peptides in bilayer membranes.     

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABA_A receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50–200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABA_A receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABA_A receptor activity and their effects on a lipid bilayer model system containing cholesterol.     

Correlation of AFM and SFA measurements concerning the stability of supported lipid bilayers

Phospholipid bilayers were studied by means of atomic force microscopy (AFM) and a surface force apparatus (SFA). The stability of the supported bilayers was described by the amount of irregularities in the topography of the membrane by means of AFM and by the occurrence of hemifusion in the SFA, which is an indicator of defective bilayers. The bilayers, composed of lipids having the same headgroup but different chain lengths in the two leaflets, were prepared by Langmuir-Blodgett deposition and transferred at different surface pressures. The topography of the supported bilayers in aqueous solution, as imaged by AFM, revealed an increasing number of defects in the supported lipid membranes with decreased deposition pressure of the outer lipid layer. These defects, which appeared in the form of monolayer and bilayer (self-assembled) thick holes within the membrane, were energetically favorable over an evenly depleted bilayer. We found that the quantity of these defects (holes of 相似文献   

The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers

We used micropipette aspiration to directly measure the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2-oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols at alcohol concentrations ranging from 0.1 to 9.8 M. The order of effectiveness in decreasing mechanical properties and increasing area per molecule was butanol>propanol>ethanol>methanol, although the lysis strain was invariant to alcohol chain-length. Quantitatively, the trend in area compressibility modulus follows Traube's rule of interfacial tension reduction, i.e., for each additional alcohol CH(2) group, the concentration required to reach the same area compressibility modulus was reduced roughly by a factor of 3. We convert our area compressibility data into interfacial tension values to: confirm that Traube's rule is followed for bilayers; show that alcohols decrease the interfacial tension of bilayer-water interfaces less effectively than oil-water interfaces; determine the partition coefficients and standard Gibbs adsorption energy per CH(2) group for adsorption of alcohol into the lipid headgroup region; and predict the increase in area per headgroup as well as the critical radius and line tension of a membrane pore for each concentration and chain-length of alcohol. The area expansion predictions were confirmed by direct measurements of the area expansion of vesicles exposed to flowing alcohol solutions. These measurements were fitted to a membrane kinetic model to find membrane permeability coefficients of short-chain alcohols. Taken together, the evidence presented here supports a view that alcohol partitioning into the bilayer headgroup region, with enhanced partitioning as the chain-length of the alcohol increases, results in chain-length-dependent interfacial tension reduction with concomitant chain-length-dependent reduction in mechanical moduli and membrane thickness.     

The influence of dioxan on the bilayer and monolayer phase transition of phospholipids

The influence of 1.4.-dioxan on the bilayer phase transition of various phospholipids was studied by differential scanning calorimetry and turbidity measurements. The addition of 1.4.-dioxan to lipid bilayers decreases the transition temperature T_m increases the transition enthalpy of the transition. The cooperativity of the transition is unaffected. The phospholipid monolayer transition from the liquid-condensed to the liquid-expanded phase was measured by recording area versus temperature curves at constant surface pressure (isobars). The monolayer transition temperature at constant surface pressure is increased when 1.4.-dioxan is added to the subphase. The change in molecular area becomes larger. A comparison of monolayer isobars on water and water/dioxan as subphase at constant surface tension rather than surface pressure leads to a decrease of the transition temperature on water/dioxan as subphase. This decrease as well as the larger change in molecular area at the monolayer transition can be correlated to the decrease in T_m and the increase in the transition enthalpy of the corresponding bilayer system. 1.4.-Dioxan seems to accumulate at the lipid head group/water interface, thus lowering the tension of the bilayer membrane. This cyclic ether can be used for altering the characteristics of bilayer membranes without disturbing the lipid chain organization.     

Differences in the modulation of collective membrane motions by ergosterol, lanosterol, and cholesterol: a dynamic light scattering study

A dynamic light scattering setup was used to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes, over a previously inaccessible range of frequency and wave number. A pure synthetic lecithin bilayer, 1,2-dielaidoyl-sn-3-glycero-phoshatidylcholine (DEPC), and binary mixtures of DEPC with 40 mol % of cholesterol, ergosterol, or lanosterol were studied. By analyzing the dynamic light scattering data (oscillation and damping curves) in terms of transverse shear motion, we extracted the lateral tension and surface viscosity of the composite bilayers for each sterol. Cholesterol gave the strongest increase in lateral tension (approximately sixfold) with respect to the DEPC control, followed by lanosterol (approximately twofold), and ergosterol (1.7-fold). Most interestingly, only cholesterol simultaneously altered the surface viscosity of the bilayer by almost two orders of magnitude, whereas the other two sterols did not affect this parameter. We interpret this unique behavior of cholesterol as a result of its previously established out-of-plane motion which allows the molecule to cross the bilayer midplane, thereby effectively coupling the bilayer leaflets to form a highly flexible but more stable composite membrane.     

Lateral tensions and pressures in membranes and lipid monolayers

The effects of lateral tension on the properties of membranes are often explained in comparison with analogous experiments on monolayers, which yield more detailed data. To calculate the effects of changes in tension on the composition of, or incorporation of amphiphiles into membranes we examine (i) the fidelity of the monolayer analogy, (ii) the range of possible tensions in a membrane, and the way in which tensions arise and (iii) the equilibrium partitioning of amphiphiles between aqueous solution and a bilayer under tension. We argue that, at the same areas per molecule, a monolayer at an $\text{n-}\text{alkane}\text{/}\text{water}$ interface is a closer analogy of the lipid bilayer than a monolayer at an air/water interface. Next, we show from a thermodynamic argument that changes in membrane tension can affect the absorption of very large amphiphiles such as proteins, but that physiological tensions are unlikely to affect the absorption of lipids or drugs. Finally we consider the possibility that the measured bulk tension in a complicated membrane such as that of the erythrocyte may be larger than the local tension in the fluid mosaic portions, and suggest a model which explains the ability of the erythrocyte membrane to withstand much higher tensions than other biological membranes and lipid bilayers.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Energetics of pore formation induced by membrane active peptides

Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.     

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.     

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.