Molecular dynamics studies of the molecular structure and interactions of cholesterol superlattices and random domains in an unsaturated phosphatidylcholine bilayer membrane

The effect of the molecular organization of lipid components on the properties of the bilayer membrane has been a topic of increasing interest. Several experimental and theoretical studies have suggested that cholesterol is not randomly distributed in the fluid-state lipid bilayer but forms nanoscale domains. Several cholesterol-enriched nanodomain structures have been proposed, including rafts, regular or maze arrays, complexes, and superlattices. At present, the molecular mechanisms by which lipid composition influences the formation and stability of lipid nanodomains remain unclear. In this study, we have used molecular dynamics (MD) simulations to investigate the effects of the molecular organization of cholesterol--superlattice versus random--on the structure of and interactions between lipids and water in lipid bilayers of cholesterol and 1-palmitoyl-2-oleoylphosphatidylcholine (cholesterol/POPC) at a fixed cholesterol mole fraction of 0.40. On the basis of four independent replicates of 200-ns MD simulations for a superlattice or random bilayer, statistically significant differences were observed in the lipid structural parameters, area per lipid, density profile, and acyl chain order profile, as well as the hydrogen bonding between various pairs (POPC and water, cholesterol and water, and POPC and cholesterol). The time evolution of the radial distribution of the cholesterol hydroxy oxygen suggests that the lateral distribution of cholesterol in the superlattice bilayer is more stable than that in the random bilayer. Furthermore, the results indicate that a relatively long simulation time, more than 100 ns, is required for these two-component bilayers to reach equilibrium and that this time is influenced by the initial lateral distribution of lipid components.     

Characterization of supported membranes on topographically patterned polymeric elastomers and their applications to microcontact printing

This article describes the fluorescence microscopy and imaging ellipsometry-based characterization of supported phospholipid bilayer formation on elastomeric substrates and its application in microcontact printing of spatially patterned phospholipid bilayers. Elastomeric stamps, displaying a uniformly spaced array of square wells (20, 50, and 100 mum linear dimensions), are prepared using poly(dimethyl)siloxane from photolithographically derived silicon masters. Exposing elastomeric stamps, following UV/ozone-induced oxidation, to a solution of small unilamellar phospholipid vesicles results in the formation of a 2D contiguous, fluid phospholipid bilayers. The bilayer covers both the elevated and depressed regions of the stamp and exhibits a lateral connectivity allowing molecular transport across the topographic boundaries. Applications of these bilayer-coated elastomeric stamps in microcontact printing of lipid bilayers reveal a fluid-tearing process wherein the bilayer in contact regions selectively transfers with 75-90% efficiency, leaving behind unperturbed patches in the depressed regions of the stamp. Next, using cholera-toxin binding fluid POPC bilayers that have been asymmetrically doped with ganglioside Gm1 ligand in the outer leaflets, we examine whether the microcontact transfer of bilayers results in the inversion of the lipid leaflets. Our results suggest a complex transfer process involving at least partial bilayer reorganization and molecular re-equilibration during (or upon) substrate transfer. Taken together, the study sheds light on the structuring of lipid inks on PDMS elastomers and provides clues regarding the mechanism of bilayer transfer. It further highlights some important differences in stamping fluid bilayers from the more routine applications of stamping in the creation of patterned self-assembled monolayers.     

Supported lipid bilayers lifted from the substrate by layer-by-layer polyion cushions on self-assembled monolayers

The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11-mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1-palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2–2.1)×10⁻⁹ cm²/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes.     

A molecular dynamics study of the lateral free energy profile of a pair of cholesterol molecules as a function of their distance in phospholipid bilayers

Free energy profile of a pair of cholesterol molecules in a leaflet of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers in the liquid-crystalline phase has been calculated as a function of their lateral distance using a combination of NPT-constant atomistic molecular dynamics calculations (P = 1 atm and T = 310.15 K) and the thermodynamic integration method. The calculated free energy clearly shows that the two cholesterol molecules form a dimer separated by a distance of 1.0-1.5 nm in POPC bilayers. Well depth of the free energy profile is about 3.5 kJ/mol, which is comparable to the thermal energy k(B)T at 310.15 K. This indicates that the aggregation of cholesterol molecules in the bilayers depends on the temperature as well as the concentration of the system. The free energy function obtained here may be used as a reference when coarse grained potential model is investigated for this two-component system. Local structure of POPC molecules around two cholesterol molecules has also been investigated.     

Lipid diffusion from single molecules of a labeled protein undergoing dynamic association with giant unilamellar vesicles and supported bilayers

It is demonstrated that single-molecule tracking of a fluorescently labeled protein undergoing transient binding to model membranes presents a useful method of obtaining fluid properties. The labeled ACBP protein was tracked during its binding to free-standing giant unilamellar vesicles (GUVs) and supported bilayers prepared from the GUVs in the same environment. The analysis of images that are blurred as a result of fast probe diffusion was discussed. An examination of the lateral diffusion trajectories revealed a homogeneous diffusion on the top segments of the GUVs with D = 6.9 +/- 0.3 microm(2)/s. The supported bilayer experiments revealed two diffusion processes, one with Df = 3.1 +/- 0.4 microm(2)/s and the other with Ds = 0.078 +/- 0.001 microm(2)/s. The 2-fold difference in the lipid bilayer mobility for the free-standing and fast components in the supported bilayers is attributed to the known effect of frictional coupling with the solid support. The slow mobile fraction in the bilayer is suggested to be associated with the migration of pore-like structures, originating from the interaction of the membrane with the glass support.     

Spectral Förster resonance energy transfer detection of protein interactions in surface-supported bilayers

F?rster resonance energy transfer (FRET), a fluorescence detection technique, is often used for sensing molecular interactions in solution and in membranes. Here we show that (1) FRET spectra can be recorded in single bilayers, supported on a surface, and (2) the fluorescein/rhodamine dye pair is an adequate reporter of FRET when spectral detection is used. Thus, measurements pertaining to molecular interactions in membranes can be carried out in supported bilayers. Spectral FRET has advantages over imaging FRET, which monitors only signal amplitudes at certain wavelength. There are also advantages to performing spectral FRET measurements in supported bilayers as compared to free liposomes in suspension. However, the spectral properties of dyes can be altered in an unexpected manner in an ordered bilayer structure on a surface, such that fluorescence detection in surface-supported bilayers is not always trivial.     

Performance of the general amber force field in modeling aqueous POPC membrane bilayers

The aim of this work was to answer the question of whether the general amber force field (GAFF) is good enough to simulate fully hydrated POPC membrane bilayers. The test system contained 128 POPC and 2985 TIP3P water molecules. The equilibration was carried out in a nonarbitrary manner to reach the stable liquid-crystalline phase. The simulations were performed by using particle mesh Ewald electrostatics implemented in molecular dynamics packages Amber8 (NPT ensembles) and NAMD2 (NPgammaT ensembles). The computational results were assessed against the following experimental membrane properties: (i) area per lipid, (ii) area compressibility modulus, (iii) order parameter, (iv) gauche conformations per acyl chain, (v) lateral diffusion coefficients, (vi) electron density profile, and (vii) bound water at the lipid/water interface. The analyses revealed that the tested force field combination approximates the experimental values at an unexpectedly good level when the NPgammaT ensemble is applied with a surface tension of 60 mN m(-1) per bilayer. It is concluded that the GAFF/TIP3P combination can be utilized for aqueous membrane bilayer simulations, as it provides acceptable accuracy for biomolecular modeling.     

Improvement of biological time-of-flight-secondary ion mass spectrometry imaging with a bismuth cluster ion source

A new liquid metal ion gun (LMIG) filled with bismuth has been fitted to a time-of-flight-secondary ion mass spectrometer (TOF-SIMS). This source provides beams of Bi(n)q+ clusters with n = 1-7 and q = 1 and 2. The appropriate clusters have much better intensities and efficiencies than the Au3+ gold clusters recently used in TOF-SIMS imaging, and allow better lateral and mass resolution. The different beams delivered by this ion source have been tested for biological imaging of rat brain sections. The results show a great improvement of the imaging capabilities in terms of accessible mass range and useful lateral resolution. Secondary ion yields Y, disappearance cross sections sigma, efficiencies E = Y/sigma , and useful lateral resolutions deltaL have been compared using the different bismuth clusters, directly onto the surface of rat brain sections and for several positive and negative secondary ions with m/z ranging from 23 up to more than 750. The efficiency and the imaging capabilities of the different primary ions are compared by taking into account the primary ion current for reasonable acquisition times. The two best primary ions are Bi3+ and Bi5(2+). The Bi3+ ion beam has a current at least five times larger than Au3+ and therefore is an excellent beam for large-area imaging. Bi5(2+) ions exhibit large secondary ions yields and a reasonable intensity making them suitable for small-area images with an excellent sensitivity and a possible useful lateral resolution <400 nm.     

Cholesterol driven alteration of the conformation and dynamics of phospholamban in model membranes

The effects of cholesterol on various membrane proteins are of long-standing interest in membrane biophysics. Here we present systematic molecular dynamics simulations (totaling 1.4 μs) of integral protein phospholamban incorporated in POPC/cholesterol bilayers (containing 0, 11.11, 22.03, 33.33, and 50 mol% of cholesterol). Phospholamban is a key regulator of cardiac contractility and has recently emerged as a potential drug target. In agreement with experiments, our results show that in a cholesterol-free pure POPC bilayer, phospholamban exhibits broad conformational distribution, ranging from the closed T-state to the extended R-state, crucial for its functionality. Increasing cholesterol concentration progressively stabilizes the bent conformers of phospholamban over open structures, and favors extensive interactions of its amphipathic N-terminal helix with the bilayer surface. The interaction energies between the N-terminal helix of PLB and different POPC/cholesterol bilayers quantitatively confirm its stronger interaction with a higher cholesterol-containing membrane. Simulation with 50 mol% of cholesterol further supports the above conclusions, where phospholamban undergoes rapid conformational transition from extended to closed form, which remains stable for the rest of the simulation time and exhibits the strongest interaction with the membrane. Cholesterol participates in hydrogen-bonding and π-stacking interactions with polar and/or aromatic residues and favors membrane association of phospholamban. We observed cholesterol-enrichment in the neighborhood of phospholamban. Moreover, as a modulator of membrane biophysical properties, cholesterol modifies the hydrophobic matching and trans-membrane tilting of phospholamban and also hinders its 2D-lateral mobility. Altogether, our results highlight atomistic details of protein-lipid interplay and provide new insights into the possible effects of cholesterol on conformational dynamics of phospholamban in membrane bilayers.     

Ultrathin spin-coated dioleoylphosphatidylcholine lipid layers in dry conditions: a combined atomic force microscopy and nanomechanical study

Atomic force microscopy (AFM) has been used to study the structural and mechanical properties of low concentrated spin-coated dioleoylphosphatidylcholine (DOPC) layers in dry environment (RH ≈ 0%) at the nanoscale. It is shown that for concentrations in the 0.1-1 mM range the structure of the DOPC spin-coated samples consists of an homogeneous lipid monolayer ～1.3 nm thick covering the whole substrate on top of which lipid bilayer (or multilayer) micro- and nanometric patches and rims are formed. The thickness of the bilayer structures is found to be ～4.5 nm (or multiples of this value for multilayer structures), while the lateral dimensions range from micrometers to tens of nanometer depending on the lipid concentration. The force required to break a bilayer (breakthrough force) is found to be ～0.24 nN. No dependence of the mechanical values on the lateral dimensions of the bilayer structures is evidenced. Remarkably, the thickness and breakthrough force values of the bilayers measured in dry environment are very similar to values reported in the literature for supported DOPC bilayers in pure water.     

Structural characterization of the voltage-sensor domain and voltage-gated K+-channel proteins vectorially oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry

The voltage-sensor domain (VSD) is a modular four-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of X-ray crystal structures for a few voltage-gated potassium (Kv) channels and a voltage-gate sodium (Nav) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e., nonconducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially oriented within a single phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) membrane investigated by X-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces, thus achieving partial to full hydration, respectively (Gupta et al. Phys. Rev. E2011, 84, 031911-1-15). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the submolecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected submolecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and exchangeable hydrogen throughout the profile structure of both the VSD itself and the VSD:POPC membrane. These two experimentally determined water and exchangeable hydrogen distribution profiles are in good agreement with molecular dynamics simulations of the VSD protein vectorially oriented within a fully hydrated POPC bilayer membrane, supporting the existence of the VSD's water pore. This approach was extended to the full-length Kv-channel (KvAP) at a solid/liquid interface, providing the separate profile structures of the KvAP protein and the POPC bilayer within the reconstituted KvAP:POPC membrane.     

Modification of Lipid Bilayer Structure by Diacylglycerol: A Comparative Study of Diacylglycerol and Cholesterol

Diacylglycerols (DAGs) are important second messengers in biomembranes, and they can activate protein kinase C and many other enzymes and receptors. However, their interactions with cholesterol and other lipids have not been previously studied using molecular dynamics (MD) simulation. In this study, nine independent atomistic MD simulations were performed to specifically investigate the interactions between di16:0DAG, 16:0,18:1-phosphatidylcholine (POPC), and cholesterol. Despite of their substantial differences in chemical structure, DAG and cholesterol produce some very similar effects in POPC bilayers: increasing acyl chain order and bilayer thickness, reducing volume-per-lipid, and decreasing lateral diffusion of molecules. More significantly, DAG also produces a strong "condensing effect" in PC bilayers. In comparison, cholesterol is more effective than DAG in producing the above effects. The driving force for the condensing effect is their molecular shape: DAG and cholesterol both have small polar headgroups and large hydrophobic bodies. In a lipid bilayer, in order to avoid the unfavorable exposure of their hydrophobic parts to water, neighboring phospholipid headgroups move toward cholesterol or DAG to provide cover. Thus, seemingly complex interactions between DAG, cholesterol and phospholipid can be clearly explained using the Umbrella Model. Our simulations confirmed the hypothesis that DAG increases the spacing between phospholipid headgroups, which is important for activating protein kinase C and other enzymes. Interestingly, our simulations also show that the conventional wisdom that the spacing created by a DAG is directly above the DAG molecule is incorrect; instead, the largest spacing usually occurs between the first and the second nearest-neighbor PC headgroups from a DAG, due to the umbrella effect.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Enhanced Lipid Diffusion and Mixing in Accelerated Molecular Dynamics

Accelerated molecular dynamics (aMD) is an enhanced sampling technique that expedites conformational space sampling by reducing the barriers separating various low-energy states of a system. Here, we present the first application of the aMD method on lipid membranes. Altogether, ～1.5 μs simulations were performed on three systems: a pure POPC bilayer, a pure DMPC bilayer, and a mixed POPC:DMPC bilayer. Overall, the aMD simulations are found to produce significant speedup in trans-gauche isomerization and lipid lateral diffusion versus those in conventional MD (cMD) simulations. Further comparison of a 70-ns aMD run and a 300-ns cMD run of the mixed POPC:DMPC bilayer shows that the two simulations yield similar lipid mixing behaviors, with aMD generating a 2-3-fold speedup compared to cMD. Our results demonstrate that the aMD method is an efficient approach for the study of bilayer structural and dynamic properties. On the basis of simulations of the three bilayer systems, we also discuss the impact of aMD parameters on various lipid properties, which can be used as a guideline for future aMD simulations of membrane systems.     

Molecular-scale structure in fluid-gel patterned bilayers: stability of interfaces and transmembrane distribution

Variations in two-dimensional membrane structures on the molecular length scale are considered to have an effect on the mechanisms by which living cell membranes maintain their functionality. We created a molecular model of a patterned bilayer to asses the static and dynamic variations of membrane lateral and transbilayer distribution in two-component lipid bilayers on the molecular level. We study DSPC (distearoylphosphatidylcholine) nanometer domains in a fluid DLPC (dilauroylphosphatidylcholine) background. The system exhibits coexisting fluid and gel phases and is studied on a microsecond time scale. We characterize three different kinds of patterns: symmetric domains, asymmetric domains, and symmetric-asymmetric domains. Preferred bilayer configurations on the nanoscale are those that minimize the hydrophobic mismatch. We find nanoscale patterns to be dynamic structures with mainly lateral and rotational diffusion affecting their stability on the microsecond time scale.     

The Role of Lateral Tension in Calcium-Induced DPPS Vesicle Rupture

We assess the role of lateral tension in rupturing anionic dipalmitoylphosphatidyserine (DPPS), neutral dipalmitoylphosphatidylcholine (DPPC), and mixed DPPS-DPPC vesicles. Binding of Ca(2+) is known to have a significant impact on the effective size of DPPS lipids and little effect on the size of DPPC lipids in bilayer structures. In the present work we utilized laser transmission spectroscopy (LTS) to assess the effect of Ca(2+)-induced stress on the stability of the DPPS and DPPC vesicles. The high sensitivity and resolution of LTS has permitted the determination of the size and shape of liposomes in solution. The results indicate a critical size after which DPPS single shell vesicles are no longer stable. Our measurements indicate Ca(2+) promotes bilayer fusion up to a maximum diameter of ca. 320 nm. These observations are consistent with a straightforward free-energy-based model of vesicle rupture involving lateral tension between lipids regulated by the binding of Ca(2+). Our results support a critical role of lateral interactions within lipid bilayers for controlling such processes as the formation of supported bilayer membranes and pore formation in vesicle fusion. Using this free energy model we are able to infer a lower bound for the area dilation modulus for DPPS (252 pN/nm) and demonstrate a substantial free energy increase associated with vesicle rupture.     

Molecular characterization of gel and liquid-crystalline structures of fully hydrated POPC and POPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of monounsaturated POPC and POPE bilayers in the gel and liquid-crystalline state at a number of temperatures, ranging from 250 to 330 K. Though the chemical structures of POPC and POPE are largely similar (choline versus ethanolamine headgroup), their transformation processes from a gel to a liquid-crystalline state are contrasting. In the similarities, the lipid tails for both systems are tilted below the phase transition and become more random above the phase transition temperature. The average area per lipid and bilayer thickness were found less sensitive to phase transition changes as the unsaturated tails are able to buffer reordering of the bilayer structure, as observed from hysteresis loops in annealing simulations. For POPC, changes in the structural properties such as the lipid tail order parameter, hydrocarbon trans-gauche isomerization, lipid tail tilt-angle, and level of interdigitation identified a phase transition at about 270 K. For POPE, three temperature ranges were identified, in which the lower one (270-280 K) was associated with a pre-transition state and the higher (290-300 K) with the post-transition state. In the pre-transition state, there was a significant increase in the number of gauche arrangements formed along the lipid tails. Near the main transition (280-290 K), there was a lowering of the lipid order parameters and a disappearance of the tilted lipid arrangement. In the post-transition state, the carbon atoms along the lipid tails became less hindered as their density profiles showed uniform distributions. This study also demonstrates that atomistic simulations of current lipid force fields are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the main transition state.     

Chemical Analysis of Inorganic and Organic Surfaces and Thin Films by Static Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

By using mass spectrometry to analyze the atomic and molecular secondary ions that are emitted from a solid surface when bombarded with ions, one obtains detailed information about the chemical composition of the surface. A time-of-flight mass spectrometer is especially suitable for the analysis of secondary ions because of its high transmission, high mass resolution, and ability to detect ions of different masses simultaneously. By using a finely focused primary ion beam it is also possible to analyze microareas and generate surface images with a lateral resolution of 0.1 μm or less. Static time-of-flight secondary ion mass spectrometry (TOF-SIMS) allows monolayer imaging and local analysis of monolayers with high sensitivity, a wide mass range, high mass resolution, and high lateral resolution. Besides information on elements and isotopes, the technique yields direct information on the molecular level and can also be used to analyze surface species of high molecular mass that are thermally unstable and cannot be vaporized. The method can be applied to practically all types of materials and sample forms, including insulators in particular. In this article the basic principles of TOF-SIMS are explained, and its analytical capabilities for both large area and imaging applications are illustrated by examples. These include silicon surfaces (both uniform and structured), thermally unstable organic molecules on surfaces, synthetic polymers, and synthetically prepared molecular surface films, particles, and fibers. Emitted neutral particles can also be analyzed by postionization with a laser, and the possibilities of this technique are discussed.     

Observing a model ion channel gating action in model cell membranes in real time in situ: membrane potential change induced alamethicin orientation change

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split α/3(10)-helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer and was measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH = 6.7 the α-helix at the N-terminus and the 3(10)-helix at the C-terminus tilt at about 72° (θ(1)) and 50° (θ(2)) versus the surface normal, respectively. When pH increases to 11.9, θ(1) and θ(2) decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ(1) for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and the bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.     

Investigations into the bending constant and edge energy of bilayers of salt-free catanionic vesicles

Using molecular dynamics simulation, we performed theoretical calculations on the curvature constant and edge energy of bilayers of salt-free, zero-charged, cationic and anionic (catanionic) surfactant vesicles composed of alkylammonium cations (C(m)(+)) and fatty acid anions (C(n)(-)). Both the minimum size and edge energy of vesicles were calculated to examine the relation between the length of the surfactant molecules and the mechanical properties of the catanionic bilayers. Our simulation results clearly demonstrate that, when the chain lengths of the cationic and anionic surfactants are equal, both the edge energy and the rigidity of the catanionic bilayers increase dramatically, changing from around 0.36 to 2.77 kBT·nm(-1) and around 0.86 to 6.51 kBT·nm(-1), respectively. For the smallest catanionic vesicles, the curvature is not uniform and the surfactant molecules adopt a multicurvature arrangement in the vesicle bilayers. We suspect that the multicurvature bending of bilayers of catanionic vesicles is a common phenomenon in rigid bilayer systems, which could aid understanding of ion transport through bilayer membranes.