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Simvastatin (Zocor) is one of the most prescribed drugs for reducing high cholesterol. Although simvastatin is ingested in its inactive lactone form, it is converted to its active dihydroxyheptanoate form by carboxylesterases in the liver. The dihydroxyheptanoate form can also be converted back to its original lactone form. Unfortunately, some of the side effects associated with the intake of simvastatin and other lipophilic statins at higher doses include statin-associated myopathy (SAM) and, in more severe cases, kidney failure. While the cause of SAM is unknown, it is hypothesized that these side effects are dependent on the localization of statins in lipid bilayers and their impact on bilayer properties. In this work, we carry out all-atom molecular dynamics simulations on both the lactone and dihydroxyheptanoate forms of simvastatin (termed "SN" and "SA", respectively) with a pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer and a POPC/cholesterol (30 mol %) binary mixture as membrane models. Additional simulations were carried out with multiple simvastatin molecules to mimic in vitro conditions that produced pleiotropic effects. Both SN and SA spontaneously diffused into the lipid bilayer, and a longer simulation time of 4 μs was needed for the complete incorporation of multiple SAs into the bilayer. By constructing potential mean force and electron density profiles, we find that SN localizes deeper within the hydrophobic interior of the bilayer and that SA has a greater tendency to form hydrogen-bonding interactions with neighboring water molecules and lipid headgroups. For the pure POPC bilayer, both SN and SA increase membrane order, while membrane fluidity increases for the POPC/cholesterol bilayer.

The lipid regulation of mammalian ion channel function has emerged as a fundamental mechanism in the control of electrical signalling and transport specificity in various cell types. In this work, we combine molecular dynamics simulations, mutagenesis, and electrophysiology to provide mechanistic insights into how lipophilic molecules (ceramide-sphingolipid probe) alter gating kinetics and K+ currents of hERG1. We show that the sphingolipid probe induced a significant left shift of activation voltage, faster deactivation rates, and current blockade comparable to traditional hERG1 blockers. Microseconds-long MD simulations followed by experimental mutagenesis elucidated ceramide specific binding locations at the interface between the pore and voltage sensing domains. This region constitutes a unique crevice present in mammalian channels with a non-swapped topology. The combined experimental and simulation data provide evidence for ceramide-induced allosteric modulation of the channel by a conformational selection mechanism., The lipid regulation of mammalian ion channel function has emerged as a fundamental mechanism in the control of electrical signalling and transport specificity. Here, the authors combine molecular dynamics simulations, mutagenesis, and electrophysiology to provide mechanistic insights into how lipophilic molecules alter gating kinetics and K+ currents of hERG1.

G protein-coupled receptors (GPCRs) are membrane-bound proteins that depend on their lipid environment to carry out their physiological function. Combined efforts from many theoretical and experimental studies on the lipid-protein interaction profile of several GPCRs hint at an intricate relationship of these receptors with their surrounding membrane environment, with several lipids emerging as particularly important. Using coarse-grained molecular dynamics simulations, we explore the lipid-protein interaction profiles of 28 different GPCRs, spanning different levels of classification and conformational states and totaling to 1 ms of simulation time. We find a close relationship with lipids for all GPCRs simulated, in particular, cholesterol and phosphatidylinositol phosphate (PIP) lipids, but the number, location, and estimated strength of these interactions is dependent on the specific GPCR as well as its conformational state. Although both cholesterol and PIP lipids bind specifically to GPCRs, they utilize distinct mechanisms. Interactions with PIP lipids are mediated by charge-charge interactions with intracellular loop residues and stabilized by one or both of the transmembrane helices linked by the loop. Interactions with cholesterol, on the other hand, are mediated by a hydrophobic environment, usually made up of residues from more than one helix, capable of accommodating its ring structure and stabilized by interactions with aromatic and charged/polar residues. Cholesterol binding to GPCRs occurs in a small number of sites, some of which (like the binding site on the extracellular side of transmembrane 6/7) are shared among many class A GPCRs. Combined with a thorough investigation of the local membrane structure, our results provide a detailed picture of GPCR-lipid interactions. Additionally, we provide an accompanying website to interactively explore the lipid-protein interaction profile of all GPCRs simulated to facilitate analysis and comparison of our data.

Specific and nonspecific protein-lipid interactions in cell membranes have important roles in an abundance of biological functions. We have used coarse-grained (CG) molecular dynamics (MD) simulations to assess lipid distributions and cholesterol flipping dynamics around surfaces in a model asymmetric plasma membrane containing one of six structurally distinct entities: aquaporin-1 (AQP1), the bacterial β-barrel outer membrane proteins OmpF and OmpX, KcsA potassium channel, WALP23 peptide, and a carbon nanotube (CNT). Our findings revealed varied lipid partitioning and cholesterol flipping times around the different solutes, and putative cholesterol binding sites in AQP1 and KcsA. The results suggest that protein-lipid interactions can be highly variable, and that surface-dependant lipid profiles are effectively manifested in CG simulations with the Martini force field.

Alloys of magnesium have been the most preferred materials in the manufacturing industry due to many attractive properties, especially high strength to weight ratio. However, outdoor applications of this metal is very scarce because of their poor wear and corrosion resistance. This work was attempted to understand the changes in the microstructure, microhardness and the wear behavior of the composite coating effected by the direct and pulsed current. Titanium carbide nano particles along with nickel metal matrix were coated on AZ80A magnesium alloy by electro codeposition technique. Special pretreatment procedures and interlayer coatings were done to prevent the metal from being corroded during electrodeposition of nickel in watts bath. The microhardness, microstructure and composition of the composite coatings were analyzed using Vicker’s microhardness tester, SEM and EDS techniques respectively. It was found that the samples coated with pulsed current exhibited more refined grains thereby yielding a compact coating with higher hardness and wear resistance in comparison with the samples coated with direct current

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

The hydrophobic surfactant proteins, SP-B and SP-C, promote rapid adsorption by the surfactant lipids to the surface of the liquid that lines the alveolar air sacks of the lungs. To gain insights into the mechanisms of their function, we used X-ray diffuse scattering (XDS) and molecular dynamics (MD) simulations to determine the location of SP-B and SP-C within phospholipid bilayers. Initial samples contained the surfactant lipids from extracted calf surfactant with increasing doses of the proteins. XDS located protein density near the phospholipid headgroup and in the hydrocarbon core, presumed to be SP-B and SP-C, respectively. Measurements on dioleoylphosphatidylcholine (DOPC) with the proteins produced similar results. MD simulations of the proteins with DOPC provided molecular detail and allowed direct comparison of the experimental and simulated results. Simulations used conformations of SP-B based on other members of the saposin-like family, which form either open or closed V-shaped structures. For SP-C, the amino acid sequence suggests a partial α-helix. Simulations fit best with measurements of XDS for closed SP-B, which occurred at the membrane surface, and SP-C oriented along the hydrophobic interior. Our results provide the most definitive evidence yet concerning the location and orientation of the hydrophobic surfactant proteins.

Band 3 is an anion exchanger for chloride/bicarbonate in the plasma membrane of erythrocytes. The function of Band 3 may be influenced by the interactions between Band 3 and lipids or lipid domains in the plasma membrane. In this work, using atomistic molecular dynamics simulation, we investigate the interactions between Band 3 and nanosized lipid domains in ternary lipid bilayers composed of saturated lipids, unsaturated lipids, and cholesterol. The simulations show asymmetric interactions between Band 3 and lipid nanodomains in the two leaflets of a neutral lipid bilayer with lower cholesterol concentration. With an increase in cholesterol concentration in the bilayer, cholesterol affects the interactions between Band 3 and lipid domains by deforming the structure of the protein. Additionally, the anionic lipids, which prefer to bind to some specific sites of Band 3, also affect the interactions between Band 3 and lipid domains. This work provides some new insight into understanding the distribution of Band 3 in the plasma membrane of erythrocytes as well as its anion exchange function.

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys122. The impacts of the AI helix on active-site dynamics and positioning of Lys122 are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface.

Aluminum 7075 has an extensive variety of utilization over different fields because of its intrinsic properties such as high ductility, high toughness, high strength and low density. It is being utilized broadly for structural parts in aviation industries to manufacture aeroplane wings, internal bearing and fuselage. In spite of having high strength to weight ratio, aluminium does not have good resistance against wear and corrosion.Surface coating technology is a well-known technique for improving the wear resistance and corrosion resistance of the coated substrate which can eliminate corrosion along with fretting and adhesive wear by providing suave surface..Generally, polyurethane (PU) coatings were done on engineering metals to provide corrosion protection; hence it is highly advantageous to coat it on aluminium 7075 to improve its corrosion resistance. Polyurethane coating technology offers excellent adhesion and corrosion resistance when compared with other traditional metal coatings such as electro plating, electroless plating, PVD, CVD etc. Polymer Matrix Composite (PMC) coatings were preferred to improve the wear resistance along with corrosion resistance on corrosive and low wear resistant metals such as copper alloys, mild steel, and aluminum alloys. In this paper, PMC coatings of polyurethane as matrix along with nano titanium carbide reinforcement were coated on Aluminium 7075 through physical deposition method. The nano TiC was blended with Epoxy polyamide (EP) and Polyurethane separately in different proportions (1%, 2% and 3%) with the help of mechanical stirrer. Epoxy polyamide along with TiC nano composite was first coated as a base coat on the Aluminium 7075 substrate at the pressure not exceeding 35bar. The polyurethane was coated as top coat along with nano titanium carbide as reinforcement on Aluminium 7075 in different volume percentage. Wear, corrosion and optical microscope studies were done. Micro hardness test reveals that the sample which was coated with 3% nano TiC along EP and 3% nano TiC & PU gives the highest hardness when compared to other samples. It is attributed to the fact that it contains the highest percentage of titanium carbide mixed to both the polyurethane and the epoxy polyamide when compared to the other samples which has nano TiC in either base coat or upper coat. Similarly, through salt spray test it was found that highest corrosion resistance was observed in the sample which was coated with nano TiC in both the EP and PU. The samples were also examined through optical microscope and test results were justified. The inclusion of nano titanium carbide to the polymer coating increases the coating hardness, wear resistance and corrosion resistance of the aluminium 7075.

P-glycoprotein contributes to multidrug resistance by exporting a broad range of substrates across the cell membrane. Using molecular dynamics simulations, Barreto-Ojeda et al. identify key lipid-binding sites and reveal lipid access pathways toward the cavity of the transporter., P-glycoprotein (P-gp) exports a broad range of dissimilar compounds, including drugs, lipids, and lipid-like molecules. Because of its substrate promiscuity, P-gp is a key player in the development of cancer multidrug resistance. Although P-gp is one of the most studied ABC transporters, the mechanism by which its substrates access the cavity remains unclear. In this study, we perform coarse-grained molecular dynamics simulations to explore possible lipid access pathways in the inward-facing conformation of P-gp embedded in bilayers of different lipid compositions. In the inward-facing orientation, only lipids from the lower leaflet access the cavity of the transporter. We identify positively charged residues at the portals of P-gp that favor lipid entrance to the cavity, as well as lipid-binding sites at the portals and within the cavity, which is in good agreement with previous experimental studies. This work includes several examples of lipid pathways for phosphatidylcholine and phosphatidylethanolamine lipids that help elucidate the molecular mechanism of lipid binding in P-gp.

Crystallographic data of the dimeric and octameric forms of fragaceatoxin C (FraC) suggested the key role of a small hydrophobic protein-protein interaction surface for actinoporins oligomerization and pore formation in membranes. However, site-directed mutagenesis studies supporting this hypothesis for others actinoporins are still lacking. Here, we demonstrate that disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity of these proteins. Our results allow for the extension of the importance of FraC protein-protein interactions in the stabilization of the oligomeric intermediates of StII and EqtII pointing out that all of these proteins follow a similar pathway of membrane disruption. These findings support the hybrid pore proposal as the universal model of actinoporins pore formation. Moreover, we reinforce the relevance of dimer formation, which appears to be a functional intermediate in the assembly pathway of some different pore-forming proteins. This article is protected by copyright. All rights reserved.

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.e., fluidity). The nitroxide probe rotational dynamics in longer chain detergents is more restricted than in shorter chain detergents, and maltoside micelles are more restricted than phosphocholine micelles. Furthermore, the micelle microviscosity can be modulated with mixtures, as demonstrated with mixtures of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate with n-dodecylphosphocholine, n-tetradecylphosphocholine, n-decyl-β-D-maltoside, or n-dodecyl-β-D-maltoside. These results indicate that observed differences in membrane protein stability in these detergents could be due to fluidity in addition to the already determined structural differences.

We report a detailed study of the structure and stability of carbohydrate-lipid interactions. Complexes of a methylmannose polysaccharide (MMP) derivative and fatty acids (FAs) served as model systems. The dependence of solution affinities and gas-phase dissociation activation energies (Ea ) on FA length indicates a dominant role of carbohydrate-lipid interactions in stabilizing (MMP+FA) complexes. Solution (1) H NMR results reveal weak interactions between MMP methyl groups and FA acyl chain; MD simulations suggest the complexes are disordered. The contribution of FA methylene groups to the Ea is similar to that of heats of transfer of n-alkanes from the gas phase to polar solvents, thus suggesting that MMP binds lipids through dipole-induced dipole interactions. The MD results point to hydrophobic interactions and H-bonds with the FA carboxyl group. Comparison of collision cross sections of deprotonated (MMP+FA) ions with MD structures suggests that the gaseous complexes are disordered.

Cholesterol is the most abundant molecule in the plasma membrane of mammals. Its distribution across the two membrane leaflets is critical for understanding how cells work. Cholesterol trans-bilayer motion (flip-flop) is a key process influencing its distribution in membranes. Despite extensive investigations, the rate of cholesterol flip-flop and its dependence on the lateral heterogeneity of membranes remain uncertain. In this work, we used atomistic molecular dynamics simulations to sample spontaneous cholesterol flip-flop events in a DPPC:DOPC:cholesterol mixture with heterogeneous lateral distribution of lipids. In addition to an overall flip-flop rate at the time scale of sub-milliseconds, we identified a significant impact of local environment on flip-flop rate. We discuss the atomistic details of the flip-flop events observed in our simulations.

Peri-operative inflammation has been extensively highlighted in cancer patients as detrimental. Treatment strategies to improve survival for cancer patients through targeting peri-operative inflammation have yet to be devised. We conducted a multi-centre, randomised controlled clinical trial using Taurolidine in non-metastatic colon cancer patients. Patients were randomly assigned to receive Taurolidine or a placebo. The primary endpoint for the study was the mean difference in day 1 IL-6 levels. Secondary clinical endpoints included rates of post-operative infections and tumor recurrence. A total of 293 patients were screened for trial inclusion. Sixty patients were randomised. Twenty-eight patients were randomised to placebo and 32 patients to Taurolidine. IL-6 levels were equivalent on day 1 post-operatively in both groups. However, IL-6 levels were significantly attenuated over the 7 day study period in the Taurolidine group compared to placebo (p = 0.04). In addition, IL-6 levels were significantly lower at day 7 in the Taurolidine group (p = 0.04). There were 2 recurrences in the placebo group at 2 years and 1 in the Taurolidine group. The median time to recurrence was 19 months in the Placebo group and 38 months in the Taurolidine group (p = 0.27). Surgical site infection was reduced in the Taurolidine treated group (p = 0.09). Peri-operative use of Taurolidine significantly attenuated circulating IL-6 levels in the initial 7 day post-operative period in a safe manner. Future studies are required to establish the impact of IL-6 attenuation on survival outcomes in colon cancer. The trial was registered with EudraCT (year = 2008, registration number = 005570–12 ) and ISRCTN (year = 2008, registration number = 77,829,558 ).

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that regulates the flow of anions across epithelia. Mutations in CFTR cause cystic fibrosis. CFTR belongs to the ATP-Binding Cassette (ABC) transporter superfamily, and gating is controlled by phosphorylation and ATP binding and hydrolysis. Recent ATP-free and ATP-bound structures of zebrafish CFTR revealed an unwound segment of transmembrane helix (TM) 8, which appears to be a unique feature of CFTR not present in other ABC transporter structures. Here, by means of 1 μs long molecular dynamics simulations, we investigate the interactions formed by this TM8 segment with nearby helices, in both ATP-free and ATP-bound states. We highlight the structural role of TM8 in maintaining the functional architecture of the pore and we describe a distinct membrane defect that is found near TM8 only in the ATP-free state. The results of the MD simulations are discussed in the context of the gating mechanism of CFTR.

Cholesterol is a key component of eukaryotic membranes, but its role in cellular biology in general and in lipid rafts in particular remains controversial. Model membranes are used extensively to determine the phase behavior of ternary mixtures of cholesterol, a saturated lipid, and an unsaturated lipid with liquid-ordered and liquid-disordered phase coexistence. Despite many different experiments that determine lipid-phase diagrams, we lack an understanding of the molecular-level driving forces for liquid phase coexistence in bilayers with cholesterol. Here, we use atomistic molecular dynamics computer simulations to address the driving forces for phase coexistence in ternary lipid mixtures. Domain formation is directly observed in a long-timescale simulation of a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, unsaturated 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, and cholesterol. Free-energy calculations for the exchange of the saturated and unsaturated lipids between the ordered and disordered phases give insight into the mixing behavior. We show that a large energetic contribution to domain formation is favorable enthalpic interactions of the saturated lipid in the ordered phase. This favorable energy for forming an ordered, cholesterol-rich phase is opposed by a large unfavorable entropy. Martini coarse-grained simulations capture the unfavorable free energy of mixing but do not reproduce the entropic contribution because of the reduced representation of the phospholipid tails. Phospholipid tails and their degree of unsaturation are key energetic contributors to lipid phase separation.

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 A resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.

Molecular dynamics (MD) simulations of membranes are often hindered by the slow lateral diffusion of lipids and the limited time scale of MD. In order to study the dynamics of mixing and characterize the lateral distribution of lipids in converged mixtures, we report microsecond-long all-atom MD simulations performed on the special-purpose machine Anton. Two types of mixed bilayers, POPE:POPG (3:1) and POPC:cholesterol (2:1), as well as a pure POPC bilayer, were each simulated for up to 2 μs. These simulations show that POPE:POPG and POPC:cholesterol are each fully miscible at the simulated conditions, with the final states of the mixed bilayers similar to a random mixture. By simulating three POPE:POPG bilayers at different NaCl concentrations (0, 0.15, and 1 M), we also examined the effect of salt concentration on lipid mixing. While an increase in NaCl concentration is shown to affect the area per lipid, tail order, and lipid lateral diffusion, the final states of mixing remain unaltered, which is explained by the largely uniform increase in Na(+) ions around POPE and POPG. Direct measurement of water permeation reveals that the POPE:POPG bilayer with 1 M NaCl has reduced water permeability compared with those at zero or low salt concentration. Our calculations provide a benchmark to estimate the convergence time scale of all-atom MD simulations of lipid mixing. Additionally, equilibrated structures of POPE:POPG and POPC:cholesterol, which are frequently used to mimic bacterial and mammalian membranes, respectively, can be used as starting points of simulations involving these membranes.

Potentials routinely used in atomistic molecular dynamics simulations are not always suitable for modeling systems at coarse-grained resolution. For example, in the calculation of traditional torsion angle potentials, numerical instability is often encountered in the case of very flexible molecules. To improve the stability and accuracy of coarse-grained molecular dynamics simulations, we propose two approaches. The first makes use of improved forms for the angle potentials: the restricted bending (ReB) potential prevents torsion angles from visiting unstable or unphysical configurations and the combined bending-torsion (CBT) potential smoothly flattens the interactions when such configurations are sampled. In the second approach, dummy-assisted dihedral (DAD), the torsion potential is applied differently: instead of acting directly on the beads, it acts on virtual beads, bound to the real ones. For simple geometrical reasons, the unstable region is excluded from the accessible conformational space. The benefits of the new approaches are demonstrated in simulations of polyethylene glycol (PEG), polystyrene (PS), and polypeptide molecules described by the MARTINI coarse-grained force field. The new potentials are implemented in an in-house version of the Gromacs package, publicly available.

Gangliosides are glycolipids in which an oligosaccharide headgroup containing one or more sialic acids is connected to a ceramide. Gangliosides reside in the outer leaflet of the plasma membrane and play a crucial role in various physiological processes such as cell signal transduction and neuronal differentiation by modulating structures and functions of membrane proteins. Because the detailed behavior of gangliosides and protein-ganglioside interactions are poorly known, we investigated the interactions between the gangliosides GM1 and GM3 and the proteins aquaporin (AQP1) and WALP23 using equilibrium molecular dynamics simulations and potential of mean force calculations at both coarse-grained (CG) and atomistic levels. In atomistic simulations, on the basis of the GROMOS force field, ganglioside aggregation appears to be a result of the balance between hydrogen bond interactions and steric hindrance of the headgroups. GM3 clusters are slightly larger and more ordered than GM1 clusters due to the smaller headgroup of GM3. The different structures of GM1 and GM3 clusters from atomistic simulations are not observed at the CG level based on the Martini model, implying a difference in driving forces for ganglioside interactions in atomistic and CG simulations. For protein-ganglioside interactions, in the atomistic simulations, GM1 lipids bind to specific sites on the AQP1 surface, whereas they are depleted from WALP23. In the CG simulations, the ganglioside binding sites on the AQP1 surface are similar, but ganglioside aggregation and protein-ganglioside interactions are more prevalent than in the atomistic simulations. Using the polarizable Martini water model, results were closer to the atomistic simulations. Although experimental data for validation is lacking, we proposed modified Martini parameters for gangliosides to more closely mimic the sizes and structures of ganglioside clusters observed at the atomistic level.

Due to antimicrobial resistance, the development of new drugs to combat bacterial and fungal infections is an important area of research. Nature uses short, charged, and amphipathic peptides for antimicrobial defense, many of which disrupt the lipid membrane in addition to other possible targets inside the cell. Computer simulations have revealed atomistic details for the interactions of antimicrobial peptides and cell-penetrating peptides with lipid bilayers. Strong interactions between the polar interface and the charged peptides can induce bilayer deformations - including membrane rupture and peptide stabilization of a hydrophilic pore. Here, we performed microsecond-long simulations of the antimicrobial peptide CM15 in a POPC bilayer expecting to observe pore formation (based on previous molecular dynamics simulations). We show that caution is needed when interpreting results of equilibrium peptide-membrane simulations, given the length of time single trajectories can dwell in local energy minima for 100's of ns to microseconds. While we did record significant membrane perturbations from the CM15 peptide, pores were not observed. We explain this discrepancy by computing the free energy for pore formation with different force fields. Our results show a large difference in the free energy barrier (ca. 40 kJ/mol) against pore formation predicted by the different force fields that would result in orders of magnitude differences in the simulation time required to observe spontaneous pore formation. This explains why previous simulations using the Berger lipid parameters reported pores induced by charged peptides, while with CHARMM based models pores were not observed in our long time-scale simulations. We reconcile some of the differences in the distance dependent free energies by shifting the free energy profiles to account for thickness differences between force fields. The shifted curves show that all the models describe small defects in lipid bilayers in a consistent manner, suggesting a common physical basis.

Helix A of bacteriorhodopsin was simulated by using molecular dynamics both in isolation and as part of the complete protein. A POPC lipid bilayer and an octane monolayer were used as model membranes. Comparison of various systems showed octane to be a good alternative to lipid bilayer membranes providing fast equilibration, increased sampling, and decreased computational cost. Similarly single-helix simulations were found to capture some of the details of the full-protein simulations. In particular, aromatic side chains were found to anchor in identical conformations in all simulations, regardless of the absence of a lipid-water interface or the remainder of the protein. Simulations displayed a remarkable robustness with respect to simulation parameters and system set up.

International audience; Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid or iduronic acid, linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine. GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix, control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior and differentiation. Niche adapted bacteria express enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. PL have been classified into 24 sequence-related families. Comparison of 3D structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level, and identified occurrences of convergent evolution. We have characterized structurally and enzymatically heparinase III from Bacteroides thetaiotaomicron (BtHepIII; gene BT4657), which is classified within the PL12 family. BtHepIII is a 72.5 kDa protein. We present the X-ray structures of two crystal forms of BtHepIII at resolution 1.8 and 2.4 angstrom. BtHepIII contains two domains, the N-terminal alpha-helical domain forming a toroid and the C-terminal beta-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases, we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of BtHepIII toward sulfate-poor heparan sulfate substrate.

Glycolipids, which are composed of oligosaccharide headgroups and glycerol or sphingosine backbones, are important components of plasma membranes. Gangliosides are glycolipids in which an oligosaccharide headgroup containing one or more sialic acids is connected to ceramide. The negatively charged headgroups of gangliosides extend out of the plasma membrane extracellular side and play a crucial role in cell-cell recognition. Since the behavior of gangliosides and their interactions with proteins are poorly known, we conducted molecular dynamics simulations at both the coarse-grained and atomistic levels. We investigated the interactions between the gangliosides GM1 and GM3 and the proteins aquaporin and WALP23. GM1 and GM3 aggregated in clusters in POPC bilayers containing 17% gangliosides. These clusters pack around the protein and peptide in the bilayer. The cluster size is much smaller in polarizable MARTINI water than in standard MARTINI water. In atomistic simulations, the large GM1 and GM3 clusters found in standard MARTINI water also fell apart to clusters of smaller size. Gangliosides and ganglioside-protein interaction free energies in POPC bilayers obtained with polarizable MARTINI water are roughly half of those with standard MARTINI water. Corresponding free energies in atomistic simulations revealed different free energy landscapes and even weaker interactions.

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

In spite of extensive research and use in medical practice, the precise molecular mechanism of volatile anesthetic action remains unknown. The distribution of anesthetics within lipid bilayers and potential targeting to membrane proteins is thought to be central to therapeutic function. Therefore, obtaining a molecular level understanding of volatile anesthetic partitioning into lipid bilayers is of vital importance to modern pharmacology. In this study we investigate the partitioning of the prototypical anesthetic, chloroform, into lipid bilayers and different organic solvents using molecular dynamics simulations with potential models ranging from simplified coarse-grained MARTINI to additive and polarizable CHARMM all-atom force fields. Many volatile anesthetics display significant inducible dipole moments, which correlate with their potency, yet the exact role of molecular polarizability in their stabilization within lipid bilayers remains unknown. We observe that explicit treatment of atomic polarizability makes it possible to accurately reproduce solvation free energies in solvents with different polarities, allowing for quantitative studies in heterogeneous molecular distributions, such as lipid bilayers. We calculate the free energy profiles for chloroform crossing lipid bilayers to reveal a role of polarizability in modulating chloroform partitioning thermodynamics via the chloroform-induced dipole moment and highlight competitive binding to the membrane core and toward the glycerol backbone that may have significant implications for understanding anesthetic action.

In recent years, atomistic molecular simulations have become a method of choice for studying the interaction of small molecules, peptides, and proteins with biological membranes. Here, we critically examine the statistical convergence of equilibrium properties in molecular simulations of two amino acid side-chain analogs, leucine and arginine, in the presence of a hydrated phospholipid bilayer. To this end, the convergence of the standard binding free energy for the reversible insertion of the solutes in the bilayer is systematically assessed by evaluating dozens of separate sets of umbrella sampling calculations for a total simulation time exceeding 400 μs. We identify rare and abrupt transitions in bilayer structure as a function of solute insertion depth. These transitions correspond to the slow reorganization of ionic interactions involving zwitterionic phospholipid headgroups when the solutes penetrate the lipid-water interface and when arginine is forced through the bilayer center. These rare events are shown to constitute hidden sampling barriers that limit the rate of convergence of equilibrium properties and result in systematic sampling errors. Our analysis demonstrates that the difficulty of attaining convergence for lipid bilayer-embedded solutes has, in general, been drastically underestimated. This information will assist future studies in improving accuracy by selecting a more appropriate reaction coordinate or by focusing computational resources on those regions of the reaction coordinate that exhibit slow convergence of equilibrium properties.

The partitioning of proteins and peptides at the membrane/water interface is a key step in many processes, including the action of antimicrobial peptides, cell-penetrating peptides, and toxins, as well as signaling. To develop a computational model that can be used to accurately represent such systems, the underlying model must be able to quantitatively represent the partitioning preferences of amino acids in the lipid membrane. The MARTINI model provides a consistent set of parameters for building coarse-grained models of systems involving lipids and proteins. Even though MARTINI is parametrized to reproduce the partitioning behavior of small molecules, its ability to reproduce partitioning preferences of amino acids at lipid/water interfaces has never been tested. In this study, we measured the partitioning free energies of side chains of amino acids using alchemical simulations and umbrella sampling. The pentapeptides of sequence Ac-WLXLL were simulated at the POPC/water and cyclohexane/water interfaces using MARTINI, and the computed free energies were compared with the Wimley-White hydrophobicity scale. The free energy values obtained using the free energy perturbation, thermodynamic integration, and umbrella sampling methods were compared to gain insight into the most efficient method and the degree of sampling required to obtain statistically accurate free energies for use with atomistic force fields in future work. With the standard MARTINI water model, the amino acids D, E, K, and R were found to be significantly too favorable in hydrophobic environments, whereas with the polarizable water model, the amino acids D, E, K, and R were found to give correct free energies of partitioning. The amino acids P and F showed significant deviations from the experimental values. This model system will be used in future improvements to the MARTINI model.

We simulated spontaneous fusion of small unilamellar vesicles mediated by lung surfactant protein B (SP-B) using the MARTINI force field. An SP-B monomer triggers fusion events by anchoring two vesicles and facilitating the formation of a lipid bridge between the proximal leaflets. Once a lipid bridge is formed, fusion proceeds via a previously described stalk – hemifusion diaphragm – pore-opening pathway. In the absence of protein, fusion of vesicles was not observed in either unbiased simulations or upon application of a restraining potential to maintain the vesicles in close proximity. The shape of SP-B appears to enable it to bind to two vesicles at once, forcing their proximity, and to facilitate the initial transfer of lipids to form a high-energy hemifusion intermediate. Our results may provide insight into more general mechanisms of protein-mediated membrane fusion, and a possible role of SP-B in the secretory pathway and transfer of lung surfactant to the gas exchange interface.

Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [gamma-(32)P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.

A material family to replace the current superalloys in aeronautical gas turbine engines is considered to be that of gamma Titanium Aluminide (-TiAl) alloys. Structural components in aeronautical gas turbine engines typically experience large variations in temperatures and multiaxial states of stress under non-isothermal conditions. The uniaxial, torsional and bi-axial thermo-mechanical fatigue (TMF) behaviour of this -TiAl alloy have been examined at 400 – 800oC with strain amplitudes from 0.15% to 0.7%. The tests were conducted at both in-phase (IP) and out-of-phase (OP). The effects of TMF on the microstructure were also investigated. For the same equivalent mechanical strain amplitude uniaxial IP tests showed significantly longer lifetimes than pure torsional TMF tests. The non-proportional multiaxial OP test showed the lowest lifetimes at the same equivalent mechanical strain amplitude compared to the other types of tests.

Lipid peroxidation plays an important role in cell membrane damage. We investigated the effect of lipid peroxidation on the properties of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) lipid bilayers using molecular dynamics simulations. We focused on four main oxidation products of linoleic acid with either a hydroperoxide or an aldehyde group: 9-trans, cis-hydroperoxide linoleic acid, 13-trans, cis-hydroperoxide linoleic acid, 9-oxo-nonanoic acid, and 12-oxo-9-dodecenoic acid. These oxidized chains replaced the sn-2 linoleate chain. The properties of PLPC lipid bilayers were characterized as a function of the concentration of oxidized lipids, with concentrations from 2.8% to 50% for each oxidation product. The introduction of oxidized functional groups in the lipid tail leads to an important conformational change in the lipids: the oxidized tails bend toward the water phase and the oxygen atoms form hydrogen bonds with water and the polar lipid headgroup. This conformational change leads to an increase in the average area per lipid and, correspondingly, to a decrease of the bilayer thickness and the deuterium order parameters for the lipid tails, especially evident at high concentrations of oxidized lipid. Water defects are observed in the bilayers more frequently as the concentration of the oxidized lipids is increased. The changes in the structural properties of the bilayer and the water permeability are associated with the tendency of the oxidized lipid tails to bend toward the water interface. Our results suggest that one mechanism of cell membrane damage is the increase in membrane permeability due to the presence of oxidized lipids.

Naturally occurring and synthetic peptides may be a novel class of clinically useful antibiotics. A large body of experimental data on structure function relationships for such peptides is available, but the molecular mechanism of their action remains elusive in most cases. Computer simulations can give detailed insights into the interactions between peptides and lipid bilayers, at least one crucial step in the antimicrobial mechanism. Here we review recent simulations of antimicrobial peptides and discuss potential future contributions of computer simulations in understanding and ultimately designing antimicrobial peptides.

Fluorescent dyes revolutionized and expanded our understanding of biological membranes. The interpretation of experimental fluorescence data in terms of membrane structure, however, requires detailed information about the molecular environment of the dyes. Nile red is a fluorescent molecule whose excitation and emission maxima depend on the polarity of the solvent. It is mainly used as a probe to study lipid microenvironments, for example in imaging the progression of damage to the myelin sheath in multiple sclerosis. In this study, we determine the position and orientation of Nile red in lipid bilayers by calculating two-dimensional Potential of Mean Force (2D-PMF) profiles in a defect-free 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and in damaged bilayers containing two mixtures of the oxidized lipid 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine and POPC. From 2D-PMF simulations we obtain positions and orientations of Nile Red corresponding to the minimum on the binding free energy surface in three different membrane environments with increasing amounts of water, mimicking damage in biological tissue. Using representative snapshots from the simulations, we use combined quantum mechanical/molecular mechanical (QM/MM) models to calculate the emission spectrum of Nile red as a function of its local solvation environment. The results of QM and QM/MM computations are in qualitative agreement with the experimentally observed shift in fluorescence for the dye moving from aqueous solution to the more hydrophobic environment of the lipid interiors. The range of the conformation dependent values of the computed absorption-emission spectra and the lack of solvent relaxation effects in the QM/MM calculations made it challenging to delineate specific differences between the intact and damaged bilayers.

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.

The nucleotide binding domains (NBDs) are the energy supplying subunits of ATP-binding cassette (ABC) proteins. They power transport by binding and hydrolyzing ATP. Tracing the pathway between different conformational states of the NBDs during ATP binding, hydrolysis, and release has, however, proven difficult. We have used molecular dynamics simulations to study the ATP-driven association of the NBDs of the maltose ABC transporter, MalK, based on the crystal structures of its open and semiopen dimers. When MgATP was introduced into the binding pockets, the semiopen dimer transitioned to a closed conformation, whereas the open dimer evolved to a semiopen state. In the absence of docked MgATP, however, the twin NBDs of both the open and semiopen starting configurations drifted further apart. Both the presence of MgATP and direct cross-interface protein-protein hydrogen bonds, primarily involving the D-loop, quite likely play a key role in initiating closure. The simulations of the MgATP-docked semiopen form indicate that completion of closure is driven mainly by cross-interface contacts between the gamma-phosphate of ATP and residues in the signature motif. Our simulations also give insight into possible interactions of MalK with the regulatory proteins MalT and enzyme IIA(glc).

SUMMARY Recent interest in reducing nitrate levels in ground water has stimulated a re-examination of the potential use of foliar applications of urea on citrus trees. We examined the penetration characteristics of urea through isolated cuticles in relation to their thickness and surface wax content in citrus (Citrus paradisi Macf.) leaves using a finite dose-diffusion system. Isolated cuticles with the greatest integrity were mounted on diffusion half-cells with the inner morphological surface in contact with citrate buffer solution. 14 C-urea in citrate buffer was applied to the outer wax-rich morphological surface and the penetration of urea was monitored by repeated sampling of the receiver solution. Urea penetration through cuticles exhibited an asymptotic curve characterised by an initial lag phase of about 10 min, a quasi-linear phase of maximum penetration rate, averaging 2% of the urea applied h ‐1 , occurred about 40 min after urea application, followed by a plateau phase during which the rate of penetration approached zero at 144 h. Total urea penetration averaged approx. 35% and tended to decrease with increased cuticle weight until the cuticles were 6 months-old. Re-hydration of the urea deposit, with water, stimulated additional penetration of about 1%; however, this was not significant. Epicuticular wax occurred as platelets that increased with leaf age. Dewaxing the cuticles significantly enhanced the maximum penetration rates by 30% of the amount applied h ‐1 and increased total penetration by 64%. This study provides valuable details on the transport of urea through isolated cuticles which is important in fine-tuning foliar urea fertilisation in citrus trees.

A commercial NOx-storage catalyst for gasoline applications containing platinum, palladium and rhodium together with Ba/CeO2/Al2O3 has been sulfated on the engine bench at 390 and 510 °C with nominal exposures of 0.3 and 1.3 g sulphur/l catalyst. Exposures of 0.3 g/l are too low to have a notable impact on the catalytic performance. At 390 °C, the sulphur is quantitatively adsorbed as opposed to 510 °C. Sulphur is mainly adsorbed at the inlet side of the catalyst thereby shielding the downstream region. Desulfation on synthetic gas bench at 700 °C leads to a removal of 50% of the sulphur. The residual sulphur is more evenly distributed along the length of the catalyst compared to the sulphur profile in the sulfated catalyst. This leads to an improvement of the NOx-activity at the inlet side and a drop in NOx-performance at the outlet side after desulfation. Results from SEM–EDX, TPD, TPR and IR suggest that the barium component is selectively poisoned by the sulphur while alumina and ceria are not affected significantly. The insufficient desulfation of the catalyst is almost entirely due to the incomplete sulphur removal from the barium sites. In the desulfated state the IR and TPD-data suggest the presence of barium carbonate species of low thermal stability together with bulk carbonate. After sulfation the IR data indicate the presence of different sulphate species, both bulk and surface phases. Thermal ageing and sulphur poisoning affect different parts of the operational temperature window of the NOx-storage catalyst. While thermal stress alone mainly affects low temperature NOx-performance, sulphur poisoning mainly reduces high temperature activity. The results of this work help to identify strategies to improve both the sulfur resistance of the catalyst and the operational conditions for a better management of the NOx-trap system.

The alignment of pyrene in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer was investigated using two different approaches, namely solid-state (2)H-NMR spectroscopy and molecular dynamics (MD) simulations. Quadrupolar splittings from (2)H-NMR spectra of deuterated pyrene-d(10) in an oriented lipid bilayer give information about the orientation of C-D bonds with respect to the membrane normal. From MD simulations, geometric information is accessible via trajectories. By defining molecular and bond order parameters, the data from MD trajectories and NMR spectra can be compared straightforwardly. To ensure that the results from both methods are comparable, parameters of the experimental and the simulation setup were chosen to be as similar as possible. From simulations, we saw that pyrene prefers a position inside the lipid membrane near the headgroups and has no tendency to diffuse from one monolayer of the membrane to the other. The results from simulation and NMR show that the normal of the molecular plane is aligned nearly perpendicular to the bilayer normal. The long axis of pyrene lies preferentially parallel to the bilayer normal within a range of +/-30 degrees . The results from the two different methods are remarkably consistent. The good agreement can be explained by the fact that the different kind of motions of a pyrene molecule are already averaged within a few nanoseconds, which is the timescale covered by the MD simulation.

We calculated the free energy of solvation of the neutral analogs of 18 amino acid side-chains (not including glycine and proline) using the OPLS all-atom force field in TIP4P water, SPC water, and cyclohexane by molecular dynamics simulation and thermodynamic integration. The average unsigned errors in the free energies of solvation in TIP4P, SPC, and cyclohexane are 4.4, 4.9, and 2.1 kJ/mol respectively. Most of the calculated hydration free energies are not favorable enough compared to experiment. The largest errors are found for tryptophan, histidine, glutamic acid, and glutamine. The average unsigned errors in the free energy of transfer from TIP4P to cyclohexane and from SPC to cyclohexane are 4.0 and 4.1 kJ/mol, respectively. The largest errors, of more than 7.5 kJ/mol, are found for histidine, glutamine, and glutamatic acid.

Peptide-membrane interactions are important for understanding the binding, partitioning, and folding of membrane proteins; the activity of antimicrobial and fusion peptides; and a number of other processes. We describe molecular dynamics simulations (10-25 ns) of two pentapeptides Ace-WLXLL (with X = Arg or Lys side chain) (White, S. H., and Wimley, W.C. (1996) Nat. Struct. Biol. 3, 842-848) in water and three different membrane mimetic systems: (i) a water/cyclohexane interface, (ii) water-saturated octanol, and (iii) a solvated dioleoylphosphatidylcholine bilayer. A salt bridge is found between the protonated Arg or Lys side chains with the carboxyl terminus at the three interfaces. In water/cyclohexane, the salt bridge is most exposed to the water phase and least stable. In water/octanol and the lipid bilayer systems, the salt bridge once formed persists throughout the simulations. In the lipid bilayer, the salt bridge is more stable when the peptide penetrates deeper into the bilayer. In one of two peptides, a cation-pi interaction between the Arg and the Trp side chains is stable in the lipid bilayer for about 15 ns before breaking. In all cases, the conformations of the peptides are restricted by their presence at the interface and can be assigned to a few major conformational clusters. Side chains facing the water phase are most mobile. In the lipid bilayer, the peptides remain in the interface area, where they overlap with the carbonyl area of the lipid bilayer and perturb the local density profile of the bilayer. The tryptophan side chain remains in the water-lipid interface, where it interacts with the lipid choline group and forms hydrogen bonds with the ester carbonyl of the lipid and with water in the interface.

The performance of the GROMOS96 parameter set 45A3 developed for aliphatic alkanes is tested on a bilayer of dipalmitoylphosphatidylcholine (DPPC) in water in the liquid-crystalline L(alpha) phase. Variants of the force-field parameter set as well as different sets of simulation conditions or simulation parameter sets are evaluated. In the case of the force-field parameters, the van der Waals constants for the non-bonded interaction of the ester carbonyl carbon and the partial charges and charge group definition of the phosphatidylcholine head group are examined. On the methodological side, different cut-off distances for the non-bonded interactions, use of a reaction-field force due to long-range electrostatic interactions, the frequency of removal of the centre of mass motion and the strength of the coupling of the pressure of the system to the pressure bath are tested. The area per lipid, as a measure of structure, the order parameters of the chain carbons, as a measure of membrane fluidity, and the translational diffusion of the lipids in the plane of the bilayer are calculated and compared with experimental values. An optimal set of simulation parameters for which the GROMOS96 parameter set 45A3 yields a head group area, chain order parameters and a lateral diffusion coefficient in accordance with the experimental data is listed.

This paper investigates the microstructural and mechanical properties of copper metal matrix composites reinforced with B 4 C and crushed sea shell particles (fabricated using powder metallurgy). In powder form, copper is widely used in structural applications. Copper also possesses very good electrical and thermal conductivity, ductility, and corrosion resistance. B 4 C is the third-hardest-known material that also possesses excellent toughness and wear resistance. Sea shells are readily available along coastal areas. Therefore, an attempt has been made in this work to investigate the feasibility of its utilization in powder metallurgy. Two batches of samples were prepared. In the first batch, the percentage of boron carbide and copper powder were varied, and seashell powder was not included. In the second batch, the percentages of B 4 C, copper, and sea shell powder were varied in order to assess the change effected by the sea shell material. The sintered samples of both batches were subjected to microstructural characterization to ascertain the homogeneous distribution of the reinforcements. The microhardness and wear resistance of all of the fabricated samples were assessed. The results confirmed that the inclusion of 2% sea shell powder (by weight) with 10% boron carbide improved the wear resistance and hardness of the composite.

The dye 9-diethylamino-5H-benzo[α]phenoxazine-5-one, commonly known as Nile Red, is a fluorescent molecule whose position of excitation and emission maxima are dependent on the polarity of the solvent. The dye is mainly used as a probe for the determination of the lipid microenvironment.In this presentation we discuss the development of a methodology for the prediction of the optical shifts of Nile Red in various lipid bilayer environments. This approach incorporates: 1) development of an improved MM model of Nile Red, 2) Potential of Mean Force (PMF) calculations to determine the orientation and position of the dye in the bilayer, 3) clustering to identify representative configurations and 4) combined QM/MM calculations to predict the spectra of each selected configuration of Nile Red in a bilayer. We will also present preliminary results highlighting the application of the newly developed methodology.

Multinanosecond molecular dynamics simulations of gramicidin A embedded in a dimyristoylphosphatidylcholine bilayer show a remarkable structural stability for both experimentally determined conformations: the head-to-head helical dimer and the double helix. Water permeability was found to be much higher in the double helical conformation, which is explained by lower hydrogen bond-mediated enthalpic barriers at the channel entrance and its larger pore size. Free-energy perturbation calculations show that the double helical structure is stabilized by the positive charges at the N termini introduced by the desformylation, whereas the helical dimer is destabilized. Together with the recent experimental observation that desformyl gramicidin conducts water hundredfold better than gramicidin, this suggests that desformyl gramicidin A predominantly occurs in the double helical conformation.

Fatty acid aggregation is important for a number of diverse applications: from origins of life research to industrial applications to health and disease. Experiments have characterized the phase behavior of oleic acid mixtures, but the molecular details are complex and hard to probe with many experiments. Coarse-grained molecular dynamics computer simulations and free energy calculations are used to model oleic acid aggregation. From random dispersions, we observe the aggregation of oleic acid monomers into micelles, vesicles, and oil phases, depending on the protonation state of the oleic acid head groups. Worm-like micelles are observed when all the oleic acid molecules are deprotonated and negatively charged. Vesicles form spontaneously if significant amounts of both neutral and negative oleic acid are present. Oil phases form when all the fatty acids are protonated and neutral. This behavior qualitatively matches experimental observations of oleic acid aggregation. To explain the observed phase behavior, we use umbrella sampling free energy calculations to determine the stability of individual monomers in aggregates compared to water. We find that both neutral and negative oleic acid molecules prefer larger aggregates, but neutral monomers prefer negatively charged aggregates and negative monomers prefer neutral aggregates. Both neutral and negative monomers are most stable in a DOPC bilayer, with implications on fatty acid adsorption and cellular membrane evolution. Although the CG model qualitatively reproduces oleic acid phase behavior, we show that an updated polarizable water model is needed to more accurately predict the shift in pKa for oleic acid in model bilayers.

The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.