Structural Properties of Inverted Hexagonal Phase: A Hybrid Computational and Experimental Approach.

Inverted/reverse hexagonal (HII) phases are of special interest in several fields of research, including nanomedicine. We used molecular dynamics (MD) simulation to study HII systems composed of dioleoylphosphatidylethanolamine (DOPE) and palmitoyloleoylphosphatidylethanolamine (POPE) at several hydration levels and temperatures. The effect of the hydration level on several HII structural parameters, including deuterium order parameters, was investigated. We further used MD simulations to estimate the maximum hydrations of DOPE and POPE HII lattices at several given temperatures. Finally, the effect of acyl chain unsaturation degree on the HII structure was studied via comparing the DOPE with POPE HII systems. In addition to MD simulations, we used deuterium nuclear magnetic resonance (2H NMR) and small-angle X-ray scattering (SAXS) experiments to measure the DOPE acyl chain order parameters, lattice plane distances, and the water core radius in HII phase DOPE samples at several temperatures in the presence of excess water. Structural parameters calculated from MD simulations are in excellent agreement with the experimental data. Dehydration decreases the radius of the water core. An increase in hydration level slightly increased the deuterium order parameter of lipids acyl chains, whereas an increase in temperature decreased it. Lipid cylinders undulated along the cylinder axis as a function of hydration level. The maximum hydration levels of PE HII phases at different temperatures were successfully predicted by MD simulations based on a single experimental measurement for the lattice plane distance in the presence of excess water. An increase in temperature decreases the maximum hydration and consequently the radius of the water core and lattice plane distances. Finally, DOPE formed HII structures with a higher curvature compared to POPE, as expected. We propose a general protocol for constructing computational HII systems that correspond to the experimental systems. This protocol could be used to study HII systems composed of molecules other than the PE systems used here and to improve and validate force field parameters by using the target data in the HII phase.

Molecular Ordering in Lipid Monolayers: An Atomistic Simulation.

We report on atomistic simulations of DPPC lipid monolayers using the CHARMM36 lipid force field (and also the Slipid force field as a control case), combined with a four-point OPC water model. The entire two-phase region where domains of the "liquid-condensed" (LC) phase coexist with domains of the "liquid-expanded" (LE) phase has been explored. The simulations are long enough that the complete phase-transition stage, with two domains coexisting in the monolayer, is reached in all cases. Also, system sizes used are larger than those in previous works. As expected, domains of the minority phase are elongated, emphasizing the importance of anisotropic van der Waals and/or electrostatic dipolar interactions in the monolayer plane. The molecular structure is quantified in terms of distribution functions for the hydrocarbon chains and the PN dipoles. In contrast to previous work, where average distributions are calculated, distributions are here extracted for each of the coexisting phases by first identifying lipid molecules that belong to either LC or LE regions. In the case of the CHARMM36 force field, the three-dimensional distributions show that the average tilt angle of the chains with respect to the normal outward direction is (39.0 ± 0.1)° in the LC phase and (48.1 ± 0.5)° in the LC phase. In the case of the PN dipoles, the distributions indicate a tilt angle of (110.8 ± 0.5)° in the LC phase and (112.5 ± 0.5)° in the LE phase. These results are quantitatively different from those in previous works, which indicated a smaller normal component of the PN dipole. Also, the distributions of the monolayer-projected chains and PN dipoles have been calculated. Chain distributions peak along a particular direction in the LC domains, while they are uniform in the LE phase. Long-range ordering associated with the projected PN dipoles is absent in both phases. These results strongly suggest that LC domains do not exhibit dipolar ordering in the plane of the monolayer, the effect of these components being averaged out at short distances. Therefore, the only relevant component of the molecular dipoles, with regard to both intra- and long-range interdomain interactions, is normal to the monolayer. Also, the local orientation of chain projections is almost constant in LC domains and points in the direction along which domains are elongated, suggesting that the line tension driving the phase transition might be anisotropic with respect to the interfacial domain boundary.

Computational and experimental approaches for investigating nanoparticle-based drug delivery systems.

Most therapeutic agents suffer from poor solubility, rapid clearance from the blood stream, a lack of targeting, and often poor translocation ability across cell membranes. Drug/gene delivery systems (DDSs) are capable of overcoming some of these barriers to enhance delivery of drugs to their right place of action, e.g. inside cancer cells. In this review, we focus on nanoparticles as DDSs. Complementary experimental and computational studies have enhanced our understanding of the mechanism of action of nanocarriers and their underlying interactions with drugs, biomembranes and other biological molecules. We review key biophysical aspects of DDSs and discuss how computer modeling can assist in rational design of DDSs with improved and optimized properties. We summarize commonly used experimental techniques for the study of DDSs. Then we review computational studies for several major categories of nanocarriers, including dendrimers and dendrons, polymer-, peptide-, nucleic acid-, lipid-, and carbon-based DDSs, and gold nanoparticles. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Interactions of key charged residues contributing to selective block of neuronal sodium channels by µ-conotoxin KIIIA.

Voltage-gated sodium channels are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias (Na(v)1.5), myotonias (Na(v)1.4), epilepsies (Na(v)1.2), and pain (Na(v)1.7). Thus, there is strong motivation to identify isoform-specific blockers and the molecular determinants underlying their selectivity among these channels. µ-Conotoxin KIIIA blocks rNa(v)1.2 (IC(50), 5 nM), rNa(v)1.4 (37 nM), and hNa(v)1.7 (97 nM), expressed in mammalian cells, with high affinity and a maximal block at saturating concentrations of 90 to 95%. Mutations of charged residues on both the toxin and channel modulate the maximal block and/or affinity of KIIIA. Two toxin substitutions, K7A and R10A, modulate the maximal block (52-70%). KIIIA-H12A and R14A were the only derivatives tested that altered Na(v) isoform specificity. KIIIA-R14A showed the highest affinity for Na(v)1.7, a channel involved in pain signaling. Wild-type KIIIA has a 2-fold higher affinity for Na(v)1.4 than for Na(v)1.7, which can be attributed to a missing outer vestibule charge in domain III of Na(v)1.7. Reciprocal mutations Na(v)1.4 D1241I and Na(v)1.7 I1410D remove the affinity differences between these two channels for wild-type KIIIA without affecting their affinities for KIIIA-R14A. KIIIA is the first µ-conotoxin to show enhanced activity as pH is lowered, apparently resulting from titration of the free N terminus. Removal of this free amino group reduced the pH sensitivity by 10-fold. Recognition of these molecular determinants of KIIIA block may facilitate further development of subtype-specific, sodium channel blockers to treat hyperexcitability disorders.

Orientation of µ-conotoxin PIIIA in a sodium channel vestibule, based on voltage dependence of its binding.

Mutant cycle analysis has been used in previous studies to constrain possible docking orientations for various toxins. As an independent test of the bound orientation of µ-conotoxin PIIIA, a selectively targeted sodium channel pore blocker, we determined the contributions to binding voltage dependence of specific residues on the surface of the toxin. A change in the "apparent valence" (zδ) of the block, which is associated with a change of a specific toxin charge, reflects a change in the charge movement within the transmembrane electric field as the toxin binds. Toxin derivatives with charge-conserving mutations (R12K, R14K, and K17R) showed zδ values similar to those of wild type (0.61 ± 0.01, mean ± S.E.M.). Charge-changing mutations produced a range of responses. Neutralizing substitutions for Arg14 and Lys17 showed the largest reductions in zδ values, to 0.18 ± 0.06 and 0.20 ± 0.06, respectively, whereas unit charge-changing substitutions for Arg12, Ser13, and Arg20 gave intermediate values (0.24 ± 0.07, 0.33 ± 0.04, and 0.32 ± 0.05), which suggests that each of these residues contributes to the dependence of binding on the transmembrane voltage. Two mutations, R2A and G6K, yielded no significant change in zδ. These observations suggest that the toxin binds with Arg2 and Gly6 facing the extracellular solution, and Arg14 and Lys17 positioned most deeply in the pore. In this study, we used molecular dynamics to simulate toxin docking and performed Poisson-Boltzmann calculations to estimate the changes in local electrostatic potential when individual charges were substituted on the toxin's surface. Consideration of two limiting possibilities suggests that most of the charge movement associated with toxin binding reflects sodium redistribution within the narrow part of the pore.

Insights into lipid-protein interactions from computer simulations.

Lipid-protein interactions play an important direct role in the function of many membrane proteins. We argue they are key players in membrane structure, modulate membrane proteins in more subtle ways than direct binding, and are important for understanding the mechanism of classes of hydrophobic drugs. By directly comparing membrane proteins from different families in the same, complex lipid mixture, we found a unique lipid environment for every protein. Extending this work, we identified both differences and similarities in the lipid environment of GPCRs, dependent on which family they belong to and in some cases their conformational state, with particular emphasis on the distribution of cholesterol. More recently, we have been studying modes of coupling between protein conformation and local membrane properties using model proteins. In more applied approaches, we have used similar methods to investigate specific hypotheses on interactions of lipid and lipid-like molecules with ion channels. We conclude this perspective with some considerations for future work, including a new more sophisticated coarse-grained force field (Martini 3), an interactive visual exploration framework, and opportunities to improve sampling.

Ionizable amino lipid interactions with POPC: implications for lipid nanoparticle function.

Lipid nanoparticles (LNPs) composed of ionizable cationic lipids are currently the leading systems for siRNA delivery in liver disease, with the major limitation of low siRNA release efficacy into the cytoplasm. Ionizable cationic lipids are known to be of critical importance in LNP structure and stability, siRNA entrapment, and endosomal disruption. However, their distribution inside the LNPs and their exact role in cytoplasmic delivery remain unclear. A recent study [Kulkarni et al., On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA, ACS Nano, 2018, 12(5), 4787-4795] on LNP-siRNA systems containing the ionizable lipid DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2 segregates from other components and forms an amorphous oil droplet in the core of LNPs. In this paper, we present evidence supporting the model proposed by Kulkarni et al. We studied KC2 segregation in the presence of POPC using molecular dynamics simulation, deuterium NMR, SAXS, and cryo-TEM experiments, and found that neutral KC2 has a high tendency to separate from POPC dispersions. KC2 confinement, upon raising the pH during the formulation process, could result in rearrangement of the internal structure of LNPs. As interactions between cationic KC2 and anionic endosomal lipids are thought to be a key factor in cargo release, KC2 confinement inside the LNP may be responsible for the observed low release efficacy.

Simulation studies of the interaction of antimicrobial peptides and lipid bilayers.

Experimental studies of a number of antimicrobial peptides are sufficiently detailed to allow computer simulations to make a significant contribution to understanding their mechanisms of action at an atomic level. In this review we focus on simulation studies of alamethicin, melittin, dermaseptin and related antimicrobial, membrane-active peptides. All of these peptides form amphipathic alpha-helices. Simulations allow us to explore the interactions of such peptides with lipid bilayers, and to understand the effects of such interactions on the conformational dynamics of the peptides. Mean field methods employ an empirical energy function, such as a simple hydrophobicity potential, to provide an approximation to the membrane. Mean field approaches allow us to predict the optimal orientation of a peptide helix relative to a bilayer. Molecular dynamics simulations that include an atomistic model of the bilayer and surrounding solvent provide a more detailed insight into peptide-bilayer interactions. In the case of alamethicin, all-atom simulations have allowed us to explore several steps along the route from binding to the membrane surface to formation of transbilayer ion channels. For those antimicrobial peptides such as dermaseptin which prefer to remain at the surface of a bilayer, molecular dynamics simulations allow us to explore the favourable interactions between the peptide helix sidechains and the phospholipid headgroups.

The mechanism of channel formation by alamethicin as viewed by molecular dynamics simulations.

Alamethicin is a 20-residue channel-forming peptide that forms a stable amphipathic alpha-helix in membrane and membrane-mimetic environments. This helix contains a kink induced by a central Gly-X-X-Pro sequence motif. Alamethicin channels are activated by a cis positive transbilayer voltage. Channel activation is suggested to correspond to voltage-induced insertion of alamethicin helices in the bilayer. Alamethicin forms multi-conductance channels in lipid bilayers. These channels are formed by parallel bundles of transmembrane helices surrounding a central pore. A change in the number of helices per bundle switches the single channel conductance level. Molecular dynamics simulations of alamethicin in a number of different environments have been used to explore its channel-forming properties. These simulations include: (i) alamethicin in solution in water and in methanol; (ii) a single alamethicin helix at the surface of a phosphatidylcholine bilayer; (iii) single alamethicin helices spanning a phosphatidylcholine bilayer; and (iv) channels formed by bundles of 5, 6, 7 or 8 alamethicin helices spanning a phosphatidylcholine bilayer. The total simulation time is c. 30 ns. Thus, these simulations provide a set of dynamic snapshots of a possible mechanism of channel formation by this peptide.

Towards the design and computational characterization of a membrane protein.

The design of a transmembrane four-helix bundle is described. We start with an idealized four-helix bundle geometry, then use statistical information to build a plausible transmembrane bundle. Appropriate residues are chosen using database knowledge on the sequences of membrane helices and loops, then the packing of the bundle core is optimized, and favorable side chain rotamers from rotamer libraries are selected. Next, we use explicit physical knowledge from biomolecular simulation force fields and molecular dynamics simulations to test whether the designed structure is physically possible. These procedures test whether the designed protein will indeed be alpha-helical, well packed and stable over a time scale of several nanoseconds in a realistic lipid bilayer environment. We then test a modeling approach that does not include sophisticated database knowledge about proteins, but rather relies on applying our knowledge of the physics that governs protein motions. This independent validation of the design is based on simulated annealing and restrained molecular dynamics simulation in vacuo, comparable to procedures used to refine NMR and X-ray structures.

Structure and dynamics of the antifungal molecules Syringotoxin-B and Syringopeptin-25A from molecular dynamics simulation.

The phytopathogen Pseudomonas syringae pv. syringae produces toxic cyclic lipodepsipeptides (CLPs): nona-peptides and syringopeptins. All CLPs inhibit the growth of many fungal species, including human pathogens, although different fungi display different degrees of sensitivity. The best studied CLPs are Syringomycin-E (SR-E), Syringotoxin-B (ST-B) and Syringopeptin-25A (SP-25A). Their biological activity is affected by membrane composition and their structural differences. We previously (Matyus et al. in Eur Biophys J 35:459-467, 2006) reported the molecular features and structural preferences of SR-E in water and octane environments. Here we investigate in atomic detail the molecular features of the two other main CLP components, ST-B and SP-25A, in water and octane by 200 ns molecular dynamics simulations (MD), using distance restraints derived from NMR NOE data (Ballio et al. in Eur J Biochem 234:747-758, 1995). We have obtained three-dimensional models of ST-B and SP-25A CLPs in different environments. These models can now be used as a basis to investigate the interactions of ST-B and SP-25A with lipid membranes an important further step towards a better understanding of the antifungal and antibacterial activity of these peptides.

Structural investigation of syringomycin-E using molecular dynamics simulation and NMR.

Syringomycin-E (SR-E) is a cyclic lipodepsinonapeptide produced by certain strains of the bacterium Pseudomonas syringae pv. syringae. It shows inhibitory effects against many fungal species, including human pathogens. Its primary biological target is the plasma membrane, where it forms channels comprised of at least six SR-E molecules. The high-resolution structure of SR-E and the structure of the channels are currently not known. In this paper, we investigate in atomic detail the molecular features of SR-E in water by NMR and in water and octane by molecular dynamics simulation (MD). We built a model of the peptide and examined its structure in water and octane in 200 ns MD simulations both with and without distance restraints derived from NMR NOE data. The resulting trajectories show good agreement with the measured NOEs and circular dichroism data from the literature and provide atomistic models of SR-E that are an important step toward a better understanding of the antifungal and antibacterial activity of this peptide.

Orientation and dynamics of benzyl alcohol and benzyl alkyl ethers dissolved in nematic lyotropic liquid crystals. 2H NMR and molecular dynamics simulations.

Most drugs have to cross cell membranes to reach their final target. A better understanding of the distribution, interactions, and dynamics of biologically active molecules in model bilayers is of fundamental importance in understanding drug functioning and design. 2H NMR quadrupole splittings (delta nu(Q)) and longitudinal relaxation times (T1) from the aromatic ring of benzyl alcohol-d5 (C0), a commonly used anesthetic, and a series of linear alkyl benzyl-d5 ethers with chain lengths from 1 to 12 carbon atoms (C1-C12), were measured. The molecules were dissolved in a nematic discotic lyotropic liquid crystal solution made of tetradecyltrimethylammonium chloride (TTAC)/decanol (DeOH)/NaCl/H2O. Values of delta nu(Q) and T1 from 1,1-dideuteriodecanol (15% enriched) and DHO (H2O with 0.2% D2O) were also measured. Delta nu(Q) of DeOH and DHO remained constant throughout the series. The value of delta nu(Q) of the para position of the ring (delta nu(p)) in C1 is 30% smaller than the delta nu(p) of C0. This is attributed to the existence of an H-bond between the alcohol hydroxyl proton and the solvent, which influences the average orientation of the ring. The relaxation data show that T1o,m is always longer than T1p and both decrease with the increase in alkyl chain length. Molecular dynamics simulations of the experimentally studied systems were performed. The aggregate was represented as a bilayer. The distribution, average orientation, and order parameters of the aromatic ring of the guest molecules in the bilayer were examined. Rotational correlation functions of all the C-D bonds and the OH bond from H2O were evaluated, allowing an estimate of the correlation times and T1. According to these results all spins relax in extreme narrowing conditions, except DeOH. Experimental and calculated T1 values differ at most by a factor of 3. However, the order of magnitude and the observed trends are well reproduced by the calculations. The aromatic ring of C0 possesses a unique average orientation in the bilayer. For the ether series, the orientation is modified and the C2 symmetry axis of the aromatic ring is exchanging between two orientations averaging the quadrupole splittings from the ortho and meta positions. The simulation supports the existence of an H-bond between C0 and the solvent not found in the ethers, which should be responsible for the observed differences.

Molecular dynamics simulation of a lipid diamond cubic phase.

This paper presents the first atomistic simulation of a cubic membrane phase. Using the molecular dynamics simulation technique both the global and the local organization of glycerolmonoolein molecules inside the diamond cubic phase are studied. Multinanosecond simulations reveal that the center of the cubic bilayer remains close to the infinite periodic minimal surface that describes the diamond geometry. We further show that the equilibrium structure of the surfactant molecules inside the cubic phase is very similar to their structure inside a simulated lamellar bilayer. The small differences arise from the packing constraints of the surfactants within the cubic phase which has an area per surfactant that increases toward the bilayer center.

A molecular dynamics study of the pores formed by Escherichia coli OmpF porin in a fully hydrated palmitoyloleoylphosphatidylcholine bilayer.

In this paper we study the properties of pores formed by OmpF porin from Escherichia coli, based on a molecular dynamics simulation of the OmpF trimer, 318 palmitoyl-oleoyl-phosphatidylethanolamine lipids, 27 Na+ ions, and 12,992 water molecules. After equilibration and a nanosecond production run, the OmpF trimer exhibits a C-alpha root mean square deviation from the crystal structure of 0.23 nm and a stable secondary structure. No evidence is found for large-scale motions of the L3 loop. We investigate the pore dimensions, conductance, and the properties of water inside the pore. This water forms a complicated pattern, even when averaged over 1 ns of simulation time. Around the pore constriction zone the water dipoles are highly structured in the plane of the membrane, oriented by the strong transversal electric field. In addition, there is a net orientation along the pore axis pointing from the extracellular to the intracellular side of the bilayer. The diffusion coefficients of water inside the pore are greatly reduced compared to bulk. We compare our results to results from model pores (Breed et al., 1996. Biophys. J. 70:1 643-1 661; Sansom et al. 1997. Biophys. J. 73:2404-241 5) and discuss implications for further theoretical work.

Voltage-dependent insertion of alamethicin at phospholipid/water and octane/water interfaces.

Understanding the binding and insertion of peptides in lipid bilayers is a prerequisite for understanding phenomena such as antimicrobial activity and membrane-protein folding. We describe molecular dynamics simulations of the antimicrobial peptide alamethicin in lipid/water and octane/water environments, taking into account an external electric field to mimic the membrane potential. At cis-positive potentials, alamethicin does not insert into a phospholipid bilayer in 10 ns of simulation, due to the slow dynamics of the peptide and lipids. However, in octane N-terminal insertion occurs at field strengths from 0.33 V/nm and higher, in simulations of up to 100 ns duration. Insertion of alamethicin occurs in two steps, corresponding to desolvation of the Gln7 side chain, and the backbone of Aib10 and Gly11. The proline induced helix kink angle does not change significantly during insertion. Polyalanine and alamethicin form stable helices both when inserted in octane and at the water/octane interface, where they partition in the same location. In water, both polyalanine and alamethicin partially unfold in multiple simulations. We present a detailed analysis of the insertion of alamethicin into the octane slab and the influence of the external field on the peptide structure. Our findings give new insight into the mechanism of channel formation by alamethicin and the structure and dynamics of membrane-associated helices.

An alamethicin channel in a lipid bilayer: molecular dynamics simulations.

We present the results of 2-ns molecular dynamics (MD) simulations of a hexameric bundle of Alm helices in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer. These simulations explore the dynamic properties of a model of a helix bundle channel in a complete phospholipid bilayer in an aqueous environment. We explore the stability and conformational dynamics of the bundle in a phospholipid bilayer. We also investigate the effect on bundle stability of the ionization state of the ring of Glu18 side chains. If all of the Glu18 side chains are ionised, the bundle is unstable; if none of the Glu18 side chains are ionized, the bundle is stable. pKA calculations suggest that either zero or one ionized Glu18 is present at neutral pH, correlating with the stable form of the helix bundle. The structural and dynamic properties of water in this model channel were examined. As in earlier in vacuo simulations (Breed et al., 1996 .Biophys. J. 70:1643-1661), the dipole moments of water molecules within the pore were aligned antiparallel to the helix dipoles. This contributes to the stability of the helix bundle.

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer.

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within a membrane protein sequence. However, these methods tend to disagree over the beginning and end residues of TM helices, posing problems for subsequent modeling and simulation studies. Molecular dynamics (MD) simulations in an explicit lipid and water environment are used to help define the TM helix of the M2 protein from influenza A virus. Based on a comparison of the results of five different secondary structure prediction algorithms, three different helix lengths (an 18mer, a 26mer, and a 34mer) were simulated. Each simulation system contained 127 POPC molecules plus approximately 3500-4700 waters, giving a total of approximately 18,000-21,000 atoms. Two simulations, each of 2 ns duration, were run for the 18mer and 26mer, and five separate simulations were run for the 34mer, using different starting models generated by restrained in vacuo MD simulations. The total simulation time amounted to 11 ns. Analysis of the time-dependent secondary structure of the TM segments was used to define the regions that adopted a stable alpha-helical conformation throughout the simulation. This analysis indicates a core TM region of approximately 20 residues (from residue 22 to residue 43) that remained in an alpha-helical conformation. Analysis of atomic density profiles suggested that the 18mer helix revealed a local perturbation of the lipid bilayer. Polar side chains on either side of this region form relatively long-lived H-bonds to lipid headgroups and water molecules.