DMPC Bilayers Research Articles

Effect of cholesterol on behavior of 5-fluorouracil (5-FU) in a DMPC lipid bilayer, a molecular dynamics study

In this work, molecular dynamics (MD) simulations were performed to investigate the effects of cholesterol on the interaction between the hydrophilic anticancer drug, 5-FU, and fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer. Several structural and dynamical parameters of DMPC bilayers with varying amounts of cholesterol (0, 25, and 50mol%) in the presence and absence of drug molecules were calculated. Moreover, the free energy barriers for translocation of one 5-FU molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). PMF studies indicated that the location of the maximum free energy barrier was in the hydrophobic middle region of bilayer, while the minimums of the barrier were located at the hydrophilic part of bilayer at the interface with water. The minimum and maximum of the free energy profiles were independent of cholesterol concentration and suggested that the drug molecules 5-FU were accumulated in the vicinity of the polar head group of lipid bilayers. Moreover, the results showed that with increasing cholesterol concentration in the bilayer, the free energy barrier for translocation of 5-FU across the bilayer also increases which can be attributed to the condensing effect of the cholesterol on the bilayer.

Caveolin in Bilayers: Can the Intramembrane U-Shaped Conformation Really Exist

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82 to S136; Cav182-136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics (MD) simulations. The secondary structure of Cav182-136 from NMR chemical shift indexing analysis serves as guideline for generating initial structural models. 50 independent MD simulations (80 ns each) are performed to identify its favorable conformation and orientation in the bilayer. Using the short simulations as a guideline, a representative configuration was chosen and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav182-136 insertion depth in the bilayer), and corroborate that Cav182-136 inserts in the membrane with a U-shaped conformation. The angle between H1 and H2 ranges from 35° to 69°, and the tilt angle of Cav182-136 is 27° ± 6°. The simulations also show that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.

Pore Formation and Dynamics of Cytolysin-A on Supported Lipid Bilayer Membranes

Ayush Agrawal1, Rajat Desikan1, Pradeep S.3, K. G. Ayappa1, J. K. Basu2, Sandhya Visweswariah41 Department of Chemical Engineering, Indian Institute of Science2 Centre for Condensed Matter Theory, Department of Physics, Indian Institute of Science3 Bioengineering program, Indian Institute of Science4 Department of Molecular Reproduction, Development and Genetics, Indian Institute of ScienceObserving the structure and dynamics of large protein assemblies on membranes has implications on understanding the general principles behind protein aggregation and engineering drug therapies. Dynamic and structural data on mesoscopic length and time scales are scarce for these systems. Cytolysin-A (ClyA) is a pore forming toxin that is known to cause unregulated pores on various cell membranes. In order to study pore formation of ClyA on model membranes, supported lipid bilayers (SLBs) of DMPC were incubated with varying concentrations of ClyA. Pore densities were quantified using atomic force microscopy (AFM) and led to the observation that a threshold toxin concentration is necessary for pore formation. The influence of the bilayer preparation protocol was observed by contrasting the pore densities on SLBs prepared using Langmuir-Blodgett (LB) deposition as well as vesicle deposition. Pore formation at varying deposition pressures on different substrates (mica, silicon) were investigated using LB prepared bilayers. Interestingly, at a high toxin concentration of 8.82 nM (large pore density regime), membrane undulations were prominently observed in the AFM images. Understanding the lipid dynamics around protein assemblies has implications on protein signalling, stability and function. Fluorescence imaging using FRAP and FCS on SLBs incubated with ClyA, and complementary molecular dynamics simulations were carried out to investigate the inherent heterogeneity in lipid dynamics in the vicinity of the ClyA pore.

Energetic View on Membrane Pore Formation

Energetic View on Membrane Pore Formation

Quantitative Characterization of Membrane Protein-Lipid Interactions

Protein-lipid interactions are important for membrane protein (MP) structure, function, and organization. Qualitatively, bilayer regulation of MP function or sorting has been under stood in terms of the hydrophobic match between MP and the lipid bilayer. However, due to difficulties in quantitative characterization of protein-lipid interactions (even in computational studies), it is difficult to understand, even on a case-by-case basis, how much and why protein stability differs from bilayer to bilayer, and how much protein-lipid interactions and a bilayer deformation penalty (caused by inclusion of the protein) contribute to such stability difference. As a first step of quantitative characterization, we calculated the transfer free energy of gramicidin A (gA) in binary lipid systems. To obtain insight into the impact of bilayer thickness and curvature on gA-lipid interactions, we have chosen two types of binary lipid systems: (1) DLPC DMPC and (2) DLPC DLPE. In order to improve sampling and accuracy of the potential of mean force (PMF), we employed window exchange umbrella sampling molecular dynamics (WEUSMD) technique with an optimal parameter set. We will discuss the preference of gA (channel) dimer to be in DLPC compared to DMPC and DLPE bilayers. For consistency check, transfer free energy calculation is required for the binary lipid system: DMPC → DLPE. Agreement will validate that the separately calculated transfer free energy profiles can be combined to characterize the changes of the gA-lipid interactions in any two chosen bilayer types. This will allow us to quantitatively map the equilibrium states of gA in bilayers with different properties and to quantitatively elucidate the consequences by the free energy decomposition method.

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.

Molecular Addressability of Lipid Membrane Embedded Calixarenes towards Cytochrome C

By means of the atomic force microscopy techniques we studied the surface topography of the supported bilayer lipid membranes (sBLM) composed of 1,2-sn-glycerodimyristoylfosfatidylcholine (DMPC) with incorporated calixarenes tOct [6]CH2COOH (CX) specific to cytochrome c (cyt c). It is supposed that cyt c interacts with CX through amino groups of lysine residues at its surface. Therefore we also applied single molecule force spectroscopy (SMFS) to analyze the mechanisms of interaction of cyt c with the CX. In later case cyt c or individual NH2 group have been connected to the AFM tip through special linker. The topography of bare sBLM in a gel state (T=19oC) revealed relatively smooth surface (RRMS=0.18 nm) and thickness ~5.1 nm which agrees well with previous studies. Incorporation of CX into DMPC bilayer resulted in increase of the surface roughness (RRMS= 0.39 nm) and in increase of thickness in average by 0.5 nm. The incubation of the layer with30 nM of cyt c resulted in a surface smoothing (RRMS=0.32 nm) and in a further increase of the thickness between 0.7 to 1.2 nm. The SMFS experiments with cyt c modified AFM tips approved its specific binding to CX and allowed us to determine the binding parameters koff (1.14 ± 0.59 s-1) and xβ (3.98 ± 0.63A). SMFS experiments with an amino-ended linker also resulted in highly specific interactions with comparable values for koff (2.74 ± 0.66 s-1) and xβ (5.91 ± 2.55 A). This suggests that both electrostatic and amino group specific interactions between cyt c and CX cavity exist.

Influence of a Central Tryptophan and of Cholesterol on the Properties of Defined Transmembrane Helical Peptides

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) provides a favorable host framework for investigations of the influence of guest amino acids, for example a third, centrally located, Trp residue, within the hydrophobic core of a well characterized transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We introduced a Trp residue into position 12 or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-W12 reveal that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation with only a low extent of dynamic averaging, comparable to GWALP23 itself. Indeed, the tilt and dynamics of GWALP23-W12 are quite similar to the values observed for GWALP23 in DMPC bilayer membranes. We have analyzed the dynamics of the peptide helices using a modified Gaussian treatment as well as a semi-static treatment. The results indicate that a central Trp residue at position 12 does not appreciably perturb the properties of bilayer-spanning GWALP23. (By contrast, Arg-12 or Lys-12, when charged, induces multi-state behavior for GWALP23 in bilayer membranes [PNAS 110, 1692].) Additionally, we are investigating the influence of cholesterol upon the properties of membrane-spanning GWALP23, GWALP23-W12 and GWALP23-K12.

The Impact of Oxidized Phospholipids on Lipid Membranes: Consequences for Mitochondrial Apoptosis

Mitochondria play a crucial role in the intrinsic apoptotic pathway. Key regulators of this pathway are the Bcl-2 family proteins who interact with the mitochondrial outer membrane to modulate membrane permeability. Intracellular oxidative stress is one major factor leading to apoptosis accompanied by the permeabilization of the mitochondrial outer membrane; a process causing release of apoptotic factors such as cytochrome c. Upon onset of intracellular stress phospholipids can become oxidized. These oxidized phospholipids (OxPls) can severely alter the properties of these mitochondrial membranes, and can therefore have i) a direct effect on the membrane properties and its perforation and ii) can have an indirect effect by altering the function of membrane-coupled Bcl-2 proteins (such as the anti-apoptotic Bcl-2 or the apoptotic Bax). We have been using either of the oxidized phospholipids (OxPls) PoxnoPC and PazePC, with an aldehyde or a carboxyl group respectively at their truncated sn-2-chain ends. Based on earlier DSC and 31P ssNMR studies of DMPC bilayers doped with either PoxnoPC or PazePC, we produced now mitochondrial outer membrane mimicking bilayers composed of POPC/POPE/TMCL where we simulated oxidative stress by incorporation of OxPls. Differential scanning calorimetry and 1H, 13C- and 31P MAS NMR at different temperatures were used to further describe the organization of these membranes and their OxPls dependent affinity to the apoptotic Bax protein, which in apoptosis becomes targeted to the mitochondria where it induces permeabilization. In addition, novel fluorescence leakage assays are used to study the influence of the presence and type of OxPls on the membrane permeabilization ability of Bax protein using giant (GUVs) and large (LUVs) unilamellar vesicles.

Modeling the Structural Properties of the Transmembrane Peptide of Influenza Hemagglutinin in a Membrane Bilayer

Like all enveloped viruses, influenza virus enters the host cell by fusing its membrane with the host membrane, in a process mediated by the hemagglutinin (HA) glycoprotein. HA is a homotrimeric protein comprised by a soluble part (which contains the receptor binding site and the fusion peptide) and a transmembrane (TM) peptide. The TM peptide attaches the protein to the viral membrane and is also thought to play a role in the fusion process. Although this peptide has been gaining considerable attention in recent years, its 3D structure and the molecular determinants of membrane insertion remain unknown.To analyze the structural determinants of membrane insertion of the TM peptide, we simulated this peptide in the presence of a DMPC bilayer [1]. We observed that the peptide adopts a mainly helical conformation and inserts in the membrane with a tilt angle of ∼64°. Simulations with mutant forms of the TM peptide revealed that mutations of Trp 24 and Tyr 5 found in the C-terminal and N-terminal regions, respectively, affect the helicity and consequently the peptide arrangement inside the membrane bilayer. Since HA is a trimer, we also performed simulations with three copies of the TM peptide embedded in a membrane. The simulations showed that the three peptides assemble in a triangular arrangement that approximately matches the positions where they should attach to the available crystallographic structure of the soluble part of HA.1. B. L. Victor, A. M. Baptista and C. M. Soares, J Chem Inf Model, 2012, 52, 3001-3012.

Influence of ceramide on the internal structure and hydration of the phospholipid bilayer studied by neutron and X-ray scattering

Small angle neutron scattering (SANS), neutron diffraction and X-ray powder diffraction were used to investigate influence of N-stearoyl phytosphingosine (CER[NP]) and α-hydroxy-N-stearoyl phytosphingosine (CER[AP]) on the internal structure and hydration of DMPC membrane in fully and partly hydrated states at T = 30 °C. Application of Fourier analysis for diffraction data and model calculations for the SANS data evidence that addition of both CER[NP] and CER[AP] in small concentrations promotes significant changes in the organization of DMPC bilayers, such as the increase of the hydrophobic core region. SANS data evidence a decrease in the average radius and polydispersity of the vesicles that can be ascribed to hydrogen bonds interactions that favor tight lipid packing with a compact, more rigid character.

Cholesterol-Diethylenetriaminepentaacetate Complexed with Thulium Ions Integrated into Bicelles To Increase Their Magnetic Alignability

Lanthanides have been used for several decades to increase the magnetic alignability of bicelles. DMPE-DTPA (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylenetriaminepentaacetate) is commonly applied to anchor the lanthanides into the bicelles. However, because DMPE-DTPA has the tendency to accumulate at the highly curved edge region of the bicelles and if located there does not contribute to the magnetic orientation energy, we have tested cholesterol-DTPA complexed with thulium ions (Tm(3+)) as an alternative chelator to increase the magnetic alignability. Differential scanning calorimetric (DSC) measurements indicate the successful integration of cholesterol-DTPA into a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayer. Cryo transmission electron microscopy and small-angle neutron scattering (SANS) measurements show that the disklike structure, that is, bicelles, is maintained if cholesterol-DTPA·Tm(3+) is integrated into a mixture of DMPC, cholesterol, and DMPE-DTPA·Tm(3+). The size of the bicelles is increased compared to the size of the bicelles obtained from mixtures without cholesterol-DTPA·Tm(3+). Magnetic-field-induced birefringence and SANS measurements in a magnetic field show that with addition of cholesterol-DTPA·Tm(3+) the magnetic alignability of these bicelles is significantly increased compared to bicelles composed of DMPC, cholesterol, and DMPE-DTPA·Tm(3+) only.

Interactions of Doxorubicin with Organized Interfacial Assemblies. 2. Spectroscopic Characterization

Doxorubicin is an anthracycline that has found wide use as a chemotherapeutic agent, with the primary limitation to its use being cardiotoxicity. Depending on the identity and location of pendent side groups, the anthracyclines exhibit varying degrees of chemotherapeutic activity and toxicity, and a key area of research activity lies in understanding how the structure of the anthracycline influences its interactions with amphiphilic interfaces. We have studied interactions between doxorubicin and interfacial adlayers of octadecylamine (C18NH2), dihexadecylphosphate (DHP), and both monolayers and bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on mica using time- and frequency-resolved spectroscopic measurements. We report surface-enhanced resonance Raman data and fluorescence lifetime and anisotropy imaging data for doxorubicin at these interfaces. For all monolayers, there is a substantial interaction between doxorubicin and the interface. For DMPC bilayers, the extent of the interaction between doxorubicin and the interface depends on how the interface was formed.

Free Energetics of Arginine Permeation into Model DMPC Lipid Bilayers: Coupling of Effective Counterion Concentration and Lateral Bilayer Dimensions

Mechanisms and underlying thermodynamic determinants of translocation of charged cationic peptides such as cell-penetrating peptides across the cellular membrane continue to receive much attention. Two widely held views include endocytotic and non-endocytotic (diffusive) processes of permeant transfer across the bilayer. Considering a purely diffusive process, we consider the free energetics of translocation of a monoarginine peptide mimic across a model DMPC bilayer. We compute potentials of mean force for the transfer of a charged monoarginine peptide unit from water to the center of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model lipid bilayer. We use fully atomistic molecular dynamics simulations coupled with the adaptive biasing force (ABF) method for free energy estimation. The estimated potential of mean force difference from bulk to bilayer center is 6.94 ± 0.28 kcal/mol. The order of magnitude of this prediction is consistent with past experimental estimates of arginine partitioning into physiological bilayers in the context of translocon-based experiments, though the correlation between the bench and computer experiments is not unambiguous. Moreover, the present value is roughly one-half of previous estimates based on all-atom molecular dynamics free energy calculations. We trace the differences between the present and earlier calculations to system sizes used in the simulations and the dependence of the contributions to the free energy from various system components (water, lipids, ions, peptide) on overall system size. By varying the bilayer lateral dimensions in simulations using only sufficient numbers of counterions to maintain overall system charge neutrality, we find the possibility of an inherent convergent transfer free energy value.

Role of the Membrane Dipole Potential for Proton Transport in Gramicidin A Embedded in a DMPC Bilayer.

The membrane potential at the water/phospholipid interfaces may play a key role for proton conduction of gramicidin A (gA). Here we address this issue by Density Functional Theory-based molecular dynamics and metadynamics simulations. The calculations, performed on gA embedded in a solvated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membrane environment (about 2,000 atoms), indicate that (i) the membrane dipole potential rises at the channel mouth by ∼0.4 V. A similar value has been measured for gA embedded in a DMPC monolayer; (ii) the calculated free energy barrier is located at the channel entrance, consistent with experiments comparing gA proton conduction in different bilayers. The electronic structures of the proton ligands (water molecules and peptide units) are similar to those in the bulk solvent. Based on these results, we suggest an important role of the membrane dipole potential for the free energy barrier of proton permeation of gA. This may provide a rationale for the large increase in the rate of proton conduction under application of a transmembrane voltage, as observed experimentally. Our calculations might suggest also a role for proton desolvation for the permeation process. This role has already emerged from EVB calculations on gA embedded in a model membrane.

Hydrogen Bonding of Cholesterol in the Lipidic Cubic Phase

The addition of cholesterol to the monoolein-based lipidic cubic phase (LCP) has been instrumental in obtaining high-resolution crystal structures of several G protein-coupled receptors. Here, we report the use of high-resolution magic angle spinning NMR spectroscopy to record and assign the isotropic (13)C chemical shifts of cholesterol in lipidic lamellar and cubic phases at different hydration levels with monoolein and chain-deuterated DMPC as host lipids. The hydrogen-bonding patterns of cholesterol in these phases were determined from the NMR data by quantum chemical calculations. The results are consistent with the normal orientation of cholesterol in lipid bilayers and with the cholesterol hydroxyl group located at the hydrophobic/hydrophilic interface. The (13)C chemical shifts of cholesterol are mostly affected by the host lipid identity with little or no dependency on the hydration (20% vs 40%) or the phase identity (lamellar vs LCP). In chain-deuterated DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers it predominately interacts with monoolein. Such differences in the hydrogen-bonding network of cholesterol may have implications for the design of experiments in monoolein-based LCP.

Simulation Study on Gold Nanoparticle-Cellular Membrane Complex in Endocytosis Process

Gold nanoparticles (AuNPs) are of biomedical importance, such as delivery vectors. Therefore, we used all-atom molecular dynamic simulations to study the interaction of AuNPs with cell membrane (DMPC bilayer). We observed that the AuNPs adhered spontaneously on the surface of the cellular membrane from the bulk phase, largely as a result of the AuNP-DMPC headgroup attraction. We calculated the potential of mean force for transferring an AuNP through a DMPC bilayer. It was observed that a high energetic barrier to AuNP was inserted into the hydrophobic core of the bilayer. The inclusion of AuNP induced local bilayer deformation and slowed fluidity of the lipid molecules in the vicinity of it. As the size of the AuNP increased, the lipids in the vicinity of the AuNP had larger local deformation and slower fluidity. We found that a nanoparticle-membrane complex was formed by the AuNP and its neighbor lipids. These neighbor lipids moved laterally together with AuNP. On average, they moved significantly more slowly than the other lipids. The nanoparticle-membrane complex not only provided a clue for endocytosis mechanism regulating the translocation of AuNPs across the cellular membrane but also helped us to better understand the endocytosis process.

Molecular Dynamics Simulations of Membrane–Sugar Interactions

It is well documented that disaccharides in general and trehalose (TRH) in particular strongly affect physical properties and functionality of lipid bilayers. We investigate interactions between lipid membranes formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and TRH by means of molecular dynamics (MD) computer simulations. Ten different TRH concentrations were studied in the range wTRH = 0-0.20 (w/w). The potential of mean force (PMF) for DMPC bilayer-TRH interactions was determined using two different force fields, and was subsequently used in a simple analytical model for description of sugar binding at the membrane interface. The MD results were in good agreement with the predictions of the model. The net affinities of TRH for the DMPC bilayer derived from the model and MD simulations were compared with experimental results. The area per lipid increases and the membrane becomes thinner with increased TRH concentration, which is interpreted as an intercalation effect of the TRH molecules into the polar part of the lipids, resulting in conformational changes in the chains. These results are consistent with recent experimental observations. The compressibility modulus related to the fluctuations of the membrane increases dramatically with increased TRH concentration, which indicates higher order and rigidity of the bilayer. This is also reflected in a decrease (by a factor of 15) of the lateral diffusion of the lipids. We interpret these observations as a formation of a glassy state at the interface of the membrane, which has been suggested in the literature as a hypothesis for the membrane-sugar interactions.

Proline Facilitates Membrane Insertion of the Antimicrobial Peptide Maculatin 1.1 via Surface Indentation and Subsequent Lipid Disordering

The role of proline in the disruption of membrane bilayer structure upon antimicrobial peptide (AMP) binding was studied. Specifically, 31P and 2H solid-state NMR and dual polarization interferometry (DPI) were used to analyze the membrane interactions of three AMPs: maculatin 1.1 and two analogs in which Pro-15 is replaced by Gly and Ala. For NMR, deuterated dimyristoylphosphatidylcholine (d54-DMPC) and d54-DMPC/dimyristoylphosphatidylglycerol (DMPG) were used to mimic eukaryotic and prokaryotic membranes, respectively. In fluid-phase DMPC bilayer systems, the peptides interacted primarily with the bilayer surface, with the native peptide having the strongest interaction. In the mixed DMPC/DMPG bilayers, maculatin 1.1 induced DMPG phase separation, whereas the analogs promoted the formation of isotropic and lipid-enriched phases with an enhanced effect relative to the neutral DMPC bilayers. In gel-phase DMPC vesicles, the native peptide disrupted the bilayer via a surface mechanism, and the effect of the analogs was similar to that observed in the fluid phase. Real-time changes in bilayer order were examined via DPI, with changes in bilayer birefringence analyzed as a function of the peptide mass bound to the bilayer. Although all three peptides decreased the bilayer order as a function of bound concentration, maculatin 1.1 caused the largest change in bilayer structure. The NMR data indicate that maculatin 1.1 binds predominantly at the surface regions of the bilayer, and both NMR and DPI results indicate that this binding leads to a drop in bilayer order. Overall, the results demonstrate that the proline at residue 15 plays a central role in the membrane interaction of maculatin 1.1 by inducing a significant change in membrane order and affecting the ability of the bilayer to recover from structural changes induced by the binding and insertion of the peptide.

Free energy of WALP23 dimer association in DMPC, DPPC, and DOPC bilayers

The MARTINI coarse-grained model is used to gain insight into the association of WALP23 helices in three different lipid membranes: DMPC, DPPC and DOPC. Potentials of mean force describing the association of two WALP23 helices embedded in different lipid bilayers indicate no barrier of association and a stabilization of more than 20kJmol−1 of the associated state relative to the fully dissociated state. Association is strongest in DMPC, followed by DPPC and DOPC. Helix–helix association appears to be enthalpically favorable in all lipid bilayers, while the entropic contribution appears favorable only in the presence of significant positive hydrophobic mismatch, in DMPC lipids. The interpretation of this requires care given the coarse-grained nature of the simulations, but the sign of the thermodynamic quantities agrees with experimental measurements on dimerization of (AALALAA)3 peptides and the observed association free energies are within the experimental range. Both protein–protein and lipid–lipid interactions appear to strongly favor protein dimerization, while the interactions between a dimer and lipid are unfavorable relative to the interactions between two separated monomers and lipids. Dimers with antiparallel orientation appear to be thermodynamically favored over parallel dimers, particularly in conditions of greater hydrophobic mismatch, but elucidating the detailed origin of this likely requires simulations of helices for which there is structural data on the dimer. We analyze 3D density, membrane order, and membrane thickness maps using new freely available analysis programs. Although these properties differ somewhat for each lipid, perturbations extend to about 1nm for lipid density, ∼2nm for ordering and ∼2.5nm for thickness. A striking feature is the appearance and extent of systematic density fluctuations around the helices.