Lipid Force Field Research Articles

Protein Kinase A (PKA) Phosphorylation of Na+/K+-ATPase Opens Intracellular C-terminal Water Pathway Leading to Third Na+-binding site in Molecular Dynamics Simulations

Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all-atom Molecular Dynamics simulations to investigate the structural consequences of phosphorylating the Na(+)/K(+)-ATPase (NKA) residue Ser(936), which is the best characterized phosphorylation site in NKA, targeted in vivo by protein kinase A (PKA). The Molecular Dynamics simulations suggest that Ser(936) phosphorylation opens a C-terminal hydrated pathway leading to Asp(926), a transmembrane residue proposed to form part of the third sodium ion-binding site. Simulations of a S936E mutant form, for which only subtle effects are observed when expressed in Xenopus oocytes and studied with electrophysiology, does not mimic the effects of Ser(936) phosphorylation. The results establish a structural association of Ser(936) with the C terminus of NKA and indicate that phosphorylation of Ser(936) can modulate pumping activity by changing the accessibility to the ion-binding site.

Recent applications and developments of charge equilibration force fields for modeling dynamical charges in classical molecular dynamics simulations

With the continuing advances in computational hardware and novel force fields constructed using quantum mechanics, the outlook for non-additive force fields is promising. Our work in the past several years has demonstrated the utility of polarizable force fields, in our hands those based on the charge equilibration formalism, for a broad range of physical and biophysical systems. We have constructed and applied polarizable force fields for small molecules, proteins, lipids, and lipid bilayers and recently have begun work on carbohydrate force fields. The latter area has been relatively untouched by force field developers with particular focus on polarizable, non-additive interaction potential models. In this review of our recent work, we discuss the formalism we have adopted for implementing the charge equilibration method for phase-dependent polarizable force fields, lipid molecules, and small-molecule carbohydrates. We discuss the methodology, related issues, and briefly discuss results from recent applications of such force fields.

Derivation and systematic validation of a refined all-atom force field for phosphatidylcholine lipids.

An all-atomistic force field (FF) has been developed for fully saturated phospholipids. The parametrization has been largely based on high-level ab initio calculations in order to keep the empirical input to a minimum. Parameters for the lipid chains have been developed based on knowledge about bulk alkane liquids, for which thermodynamic and dynamic data are excellently reproduced. The FFs ability to simulate lipid bilayers in the liquid crystalline phase in a tensionless ensemble was tested in simulations of three lipids: 1,2-diauroyl-sn-glycero-3-phospocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phospcholine (DPPC). Computed areas and volumes per lipid, and three different kinds of bilayer thicknesses, have been investigated. Most importantly NMR order parameters and scattering form factors agree in an excellent manner with experimental data under a range of temperatures. Further, the compatibility with the AMBER FF for biomolecules as well as the ability to simulate bilayers in gel phase was demonstrated. Overall, the FF presented here provides the important balance between the hydrophilic and hydrophobic forces present in lipid bilayers and therefore can be used for more complicated studies of realistic biological membranes with protein insertions.

Molecular Dynamics Simulations of Membrane Proteins: Getting the Details Right

Over the past decade atomistic molecular dynamics simulations have become an established tool for studying the conformational dynamics and interactions with local environment of membrane proteins. While a great deal of valuable, molecular-level insight has been obtained from such simulations, in order to fully utilise their predictive power, it is important to continually validate and improve the methods and models that are used.The accuracy of molecular dynamics simulations is dependent upon the quality of the force fields used to describe the interactions between particles in the system. Whilst numerous studies have compared different atomistic protein force fields, there have been fewer studies comparing force fields for membranes/membrane protein simulations. Thus it is timely to initiate such a study.In the present work, we have tested the accuracy of five atomistic force fields used to simulate two different phospholipid membranes (namely the zwitterionic DPPC and POPC lipids). Multiple simulations, each 200 ns in length, have been performed to evaluate the reproduction of a range of physical properties. In addition, we have performed simulations of six different membrane proteins (3 alpha-helical and 3 beta-barrel proteins of varying size: melittin, KcsA, mitochondrial ADP/ATP carrier, OmpA, OmpG and FhuA) in both DPPC and POPC membranes using the same five lipid force fields, combined with appropriate protein force fields. Our simulations, which are in total over 60 microseconds in length, allow for a systematic comparison between frequently used combinations of lipid and protein force fields and thus will be a valuable resource for the membrane protein simulation community.

Molecular Dynamics Simulations of E. Coli Lipoplysacharide Bilayers

Lipopolysaccharide (LPS), a component of bacterial outer membranes, is responsible for the toxicity of Gram-negative bacteria. LPS is made up of three regions: the O-antigen, the core, and the innermost lipid A. These three components have been sequenced individually but never as one construct. Utilizing the latest C36 CHARMM lipid and carbohydrate force field, we have constructed a model of an entire E. coli LPS molecule. This strain's LPS contains a bisphosphorylated hexaacyl lipid A bound to an R1-type core. Various explicit bilayers were built of this LPS molecule with varying lengths of 0, 5, 10, and 20 repeating O6 antigen units; a single unit of O6 antigen contains 5 sugars. The simulation results of each system will be discussed in terms of per-lipid surface area, glycosidic angle distribution, O-antigen orientation, and distribution of each system component such as lipid A, R1 core, O6-antigen unit, water, and ions.

Molecular Dynamics Simulation, 31P-NMR Spectroscopy, and Microelectrophoretic Studies of Cardiolipin Bilayers

Molecular dynamics (MD) simulations of tetramyristoyl cardiolipin (TMCL) and tetraoleoyl cardiolipin (TOCL) were carried out with the newly developed CHARMM C36 lipid force field (FF), and with head group charges q= −1 and −2. The surface areas per lipid, AL, for q=-2 are 127.1 ± 0.4 A2 for TOCL and 113.2 ± 0.2 A2 for TMCL. To validate the simulation results, 70% TOCL + 30% POPC bilayer was prepared both in simulation and NMR experiment. The order parameters of POPC from simulations were higher than those from experiment. Sodium-lipid van der Waals interactions were slightly reduced through CHARMM NBFIX. This change of parameters led to 5% increase in surface area/lipid, and substantially improved agreement with experiment for q = -2 TOCL. The net charge of cardiolipin at neutral pH has been the subject of debate. Microelectrophoresis and 31P-NMR spectroscopy experiments were carried out, and indicate that the net charge is −2.

Performance of C36 Lipid Force Field in Pure and Mixed Lipid Bilayer Systems at Different Temperatures

A lipid membrane modifies its composition in response to environmental changes. Acyl chain length, ratio of saturation/unsaturation, and head group type affect how the composition of the lipid bilayer changes. The aim of this work is to examine the performance of the C36 lipid force field in pure and mixed lipid bilayer systems at different temperatures. We have performed molecular dynamics (MD) simulations of thirty-four lipid systems with ten different fatty acids and five different head groups. Nine additional systems have been simulated at slightly higher temperatures. To examine the changes of mixed lipid bilayer properties, we have simulated twenty lipid systems with pure POPC, pure DOPC, pure DPPC, mixtures of DOPC and DPPC, and mixtures of DOPC, DPPC, and POPC. These systems were simulated at four different temperatures: 283K, 293K, 303K, and 313K. The ratios of lipids in mixed bilayer systems differ according to their temperatures. We performed three independent runs for each system at each temperature to improve the conformational sampling and the statistics of the results. We will present per-lipid surface areas (SAs) and deuterium order parameters (Scd) with different head groups, different acyl chain length, and different levels of chain unsaturation at a given temperature. We will also present the influence of temperature and extent of mixture on the SAs and Scd.

Update of the Cholesterol Force Field Parameters in CHARMM

A modification of the CHARMM36 lipid force field (C36) for cholesterol, henceforth, called C36c, is reported. A fused ring compound, decalin, was used to model the steroid section of cholesterol. For decalin, C36 inaccurately predicts the heat of vaporization (~10 kJ/mol) and molar volume (~10 cc/mol), but C36c resulted in near perfect comparison with experiment. MD simulations of decalin and heptane at various compositions were run to estimate the enthalpy and volumes of mixing to compare to experiment for this simple model of cholesterol in a chain environment. Superior estimates for these thermodynamic properties were obtained with C36c versus C36. These new parameters were applied to cholesterol, and quantum mechanical calculations were performed to modify the torsional potential of an acyl chain torsion for cholesterol. This model was tested through simulations of DMPC/10% cholesterol, DMPC/30% cholesterol, and DOPC/10% cholesterol. The C36 and C36c results were similar for surface areas per lipid, deuterium order parameters, electron density profiles, and atomic form factors and generally agree well with experiment. However, C36 and C36c produced slightly different cholesterol angle distributions with C36c adopting a more perpendicular orientation with respect to the bilayer plane. The new parameters in the C36c modification should enable more accurate simulations of lipid bilayers with cholesterol, especially for those interested in the free energy of lipid flip/flop or transfer of phospholipids and/or cholesterol.

CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling.

Monosaccharide derivatives such as xylose, fucose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GlaNAc), glucuronic acid, iduronic acid, and N-acetylneuraminic acid (Neu5Ac) are important components of eukaryotic glycans. The present work details development of force-field parameters for these monosaccharides and their covalent connections to proteins via O-linkages to serine or threonine sidechains and via N-linkages to asparagine sidechains. The force field development protocol was designed to explicitly yield parameters that are compatible with the existing CHARMM additive force field for proteins, nucleic acids, lipids, carbohydrates, and small molecules. Therefore, when combined with previously developed parameters for pyranose and furanose monosaccharides, for glycosidic linkages between monosaccharides, and for proteins, the present set of parameters enables the molecular simulation of a wide variety of biologically-important molecules such as complex carbohydrates and glycoproteins. Parametrization included fitting to quantum mechanical (QM) geometries and conformational energies of model compounds, as well as to QM pair interaction energies and distances of model compounds with water. Parameters were validated in the context of crystals of relevant monosaccharides, as well NMR and/or x-ray crystallographic data on larger systems including oligomeric hyaluronan, sialyl Lewis X, O- and N-linked glycopeptides, and a lectin:sucrose complex. As the validated parameters are an extension of the CHARMM all-atom additive biomolecular force field, they further broaden the types of heterogeneous systems accessible with a consistently-developed force-field model.

Development of the CHARMM Force Field for Lipids

The development of the CHARMM additive all-atom lipid force field (FF) is traced from the early 1990's to the most recent version (C36) published in 2010. Though simulations with early versions yielded useful results, they failed to reproduce two important quantities: a zero surface tension at the experimental bilayer surface area, and the signature splitting of the deuterium order parameters in the glycerol and upper chain carbons. Systematic optimization of parameters based on high level quantum mechanical data and free energy simulations have resolved these issues, and bilayers with a wide range of lipids can be simulated in tensionless ensembles using C36. Issues associated with other all-atom lipid FFs, success and limitations in the C36 FF and ongoing developments are also discussed.

Membrane Pore Formation Induced by Acetylated and Polyethylene Glycol-Conjugated Polyamidoamine Dendrimers

We performed molecular dynamics (MD) simu-lations of 36 copies of unmodified (charged), acetylated, and polyethylene glycol (PEG)-conjugated G4 dendrimers in dimyristoylphosphatidylcholine (DMPC) bilayers with explicit water using coarse-grained (CG) lipid and PEG force fields (FF). Attachment of small PEG chains to the dendrimer leads to the same reduction in membrane permeability as does attachment of acetyl groups, while a larger PEG size or a higher degree of PEGylation induces even fewer pores. This indicates that PEGylation is more efficient than acetylation in reducing membrane permeability and cytotoxicity, in qualitative agreement with experimental findings (Kim et al. Bioconjugate Chem. 2008, 19, 1660). Attachment of larger PEG chains makes the dendrimer−PEG complex larger and more spherical. Although a larger size and a more spherical shape are usually conducive to pore formation, a thick PEG layer on the dendrimer surface blocks the charge interaction between cationic dendrimer terminals and a...

Properties of Lipid a Bilayers Analyzed by Molecular Dynamics Simulations

Lipopolysaccharide (LPS), responsible for the toxicity of Gram-negative bacteria, is made up of three regions: the O antigen, the core, and the innermost lipid A. Lipid A is the lipid component whose hydrophobic nature allows LPS to anchor to the bacteria outer membrane. Utilizing the latest C36 CHARMM lipid and carbohydrate force field, we have constructed a lipid A molecule of E. coli. Various bilayer systems were then constructed six different conditions: 1) CaCl2 at 80% water and at 303K or 323K, 2) KCl at 80% water at 303K or 323K, and 3) KCl at 40% water at 303K or 323K. We are currently running molecular dynamics simulations of these systems. The simulation results will be discussed in terms of how different temperatures, ratios of water molecules to lipids, and charged ions affects the lipid A bilayer properties such as surface area, ion location, chain order, bilayer thickness and shape, and the carbohydrate conformations. The results from these simulations will be also compared with available experimental data. This work will be the basis of the future simulations of the complex glycolipid simulations such as LPS, and membrane protein simulations in more native bilayer environments.

In Silico Model Escherichia Coli Membranes: Simulating a Lipid with a Cyclopropane Ring

Bacterial membranes are composed mostly of lipids with phosphoethanolamine (PE) and phosphoglycerol (PG) head groups and a variety of fatty acid chains. A defining characteristic of bacterial membranes is the presence of lipids with a cyclic-containing chain. To our knowledge, a phospholipid with such a chain has never been studied in a molecular dynamics simulation. Thus, novel force field parameters to describe a cyclopropane moiety were developed using parameters from the CHARMM36 (C36) general force field as a basis and high-level quantum mechanical energies. Two simple membranes and one complex membrane for use as realistic model Escherichia coli (E. coli) cytoplasmic membranes were designed for future work with secondary active transporters expressed in E. coli. Compositions of the membranes were based on several different experimental methods using an E. coli K12 strain grown on Luria broth. One simple membrane consisted of 85 % PE 18:1/16:0 (POPE) and 15 % PG 18:1/16:0(POPG), and the other of only PE cy17:0/16:0 (PMPE). The complex membrane consisted of six different phospholipids, the most prevalent being the cyclic-containing lipid, PMPE. NPT simulations were carried out at 310 K for 50 ns using the C36 lipid force field and the developed cyclopropane moiety force field for each membrane. NMR deuterium order parameters (SCD), density profiles, and diffusion constants are compared with experiment. The model membranes will provide a basis to study membrane bound proteins or other bound molecules expressed in E. coli with realistic molecular dynamic simulations.

Computational Characterization of DMPI and DMPI(4)P Head Groups

Results from Molecular Dynamics simulations of dimyristoyl-phosphatidylinositol (DMPI) using the C36 CHARMM lipid force field are compared with those from neutron-diffraction and 2H-NMR studies. Calculated and experimental quadrapolar splitting ratios and distances from the bilayer center to inositol deuteration sites are in good agreement. The simulations yield a large number of conformations consistent with the flexibility of the torsion angles between the glycerol and phosphate; the interpretation from diffraction indicated that the inositol ring tilt is within 30 degrees from parallel to the membrane normal, whereas the inositol ring in the simulations is within 40 degrees from perpendicular to the membrane normal, even pointing into the membrane. The results pave the way for the study of interactions between the phosphoinositides in lipid bilayers and their binding partners.

Molecular Dynamics Simulation Studies of Cardiolipin Bilayers

Molecular dynamics (MD) simulations of tetramyristoyl cardiolipin (TMCL) and tetraoleoyl cardiolipin (TOCL) were carried out with the newly developed CHARMM lipid force field (FF), C36, and with head group charges q= −1 and −2. The surface areas per lipid, AL, for q=-1 are 126 ± 0.1 Å2 for TOCL and 111 ± .1 Å2 for TMCL. These are 1.8 times than those of the diacyl equivalents: 63 Å2 for dimyristoylphosphatidylcholine (DMPC) at 328K, and 69 Å2 for dioleoylphosphatidylcholine (DOPC) at 310K. Area compressibility, Ka, of TOCL equals 340 ± 40 dyn/cm, approximately 50% higher than experimentally obtained for DOPC (and most diacyl lipids); an experimental value for Ka for cardiolipins is not presently available. The areas and compressibilities for TOCL from the present simulation differ substantially from those obtained by Dahlberg and Maliniak using the FF of Berger et al. under the same conditions (AL= 99 Å2; Ka = 1100 dyn/cm). The origin of the differences appears to be in the ion binding to the surfaces of the cardiolipin bilayer. Under FF of Berger et al., ions bind closer to the carbonyl group in the lipid chain region whereas C36 CHARMM FF results ion binding closer to the negatively charged phosphate groups of the head group. Deuterium order parameter measurements are underway to determine which FF yields areas more representative of the fluid state of cardiolipin bilayers.

A Novel United-Atom Force Field for Phosphatidylglycerols

Phosphatidylglycerols (PG)s are anionic lipids abundant both in prokaryotic membranes and in the chloroplast membrane of plants. In humans and animals PG lipids occur in minor amounts in pulmonary lung surfactants, blood cells and mitochondrial membranes. Similar to other negatively-charged phospholipids, PG has a very complex phase-behaviour: the phase transitions and structural organization of PG are affected both by temperature, pH, and concentration of mono- and divalent cations. Furthermore, the glycerol moiety of the PG headgroup has a complex conformational space in aqueous media, because of the presence of vicinal hydroxyl groups that are capable of of stabilizing various conformers through hydrogen bonds (H-bonds). In this work a novel united-atom force field is constructed for PG lipids as a part of the ongoing development of a large, consistent lipid force field library. The torsional and partial atomic charge parameters were calculated based on high-level ab initio quantum mechanical (QM) calculations with semiempirical molecular mechanics (MM) studies. The Lennard-Jones parameters were taken from the OPLS-UA force field developed by Jorgensen [1]. The QM and MM simulations were combined with experimental thermodynamic data of glycerol as target data for parameter optimization. The parameters were further optimized to reproduce the structural, dynamic and elastic properties of pure DMPG and POPG lipid bilayers. [1] W. L. Jorgensen, J. Phys. Chem. 1986, 90, 1276–1284.

Refining and Testing CHARMM Lipid Parameters for Biologically Important Membranes

Biological membranes form a barrier to protect the cell from its environment and consist of a wide variety of lipids. A recent modification to the CHARMM lipid force filed, CHARMM36 (C36), resulted in significant improvements in deuterium order parameters (SCD), water hydration, and area compressiblities. Moreover, C36 simulations resulted in excellent surface areas per lipid compared to experiment with NPT molecular dynamics (MD). We have extended this force field to aliphatic chain branched lipids, which exists in many bacterial membranes. MD simulations of DPhPC agree with experimental form factors and suggest that current lipid force field parameters are valid for these branched chain lipids. Branching leads to an increase in average surface areas per lipid, area elastic moduli and lipid axial relaxation times. However, MD simulations of mixed bilayers with cholesterol and polyunsaturated lipids suggest that the atomistic force field describing these lipids requires modification (improper sterol orientation and head-to-head group spacings). MD simulations on decalin resulted in a heat of vaporization that is 10 kJ/mol lower than experiment, but modifications to the Lennard-Jones parameters of cholesterol yielded near perfect agreement with experiment. Simulations with these parameters were in excellent agreement with experimental SCDs for the DMPC acyl chain and SCDs for ring and chain positions on cholesterol. Moreover, these simulations agree favorably with experimental form factors of DMPC-cholesterol bilayers. However, simulations with a polyunsaturated lipid, DAPC, and cholesterol still resulted in the abovementioned inaccuracies. Since the thickness of these polyunsaturated bilayers was too large by ∼8 A, high-level quantum mechanical (QM) calculations were used for dihedrals adjacent to the double bonds. It was found that the previous force field restricted the conformational flexibility and MD simulations are underway to test the QM-based potentials accuracy compared to experimental diffraction studies.

Combination of the CHARMM27 force field with united‐atom lipid force fields

Computer simulations offer a valuable way to study membrane systems, from simple lipid bilayers to large transmembrane protein complexes and lipid-nucleic acid complexes for drug delivery. Their accuracy depends on the quality of the force field parameters used to describe the components of a particular system. We have implemented the widely used CHARMM22 and CHARMM27 force fields in the GROMACS simulation package to (i) combine the CHARMM22 protein force field with two sets of united-atom lipids parameters; (ii) allow comparisons of the lipid CHARMM27 force field with other lipid force fields or lipid-protein force field combinations. Our tests do not show any particular issue with the combination of the all-atom CHARMM22 force field with united-atoms lipid parameters, although pertinent experimental data are lacking to assess the quality of the lipid-protein interactions. The conversion utilities allow automatic generation of GROMACS simulation files with CHARMM nucleic acids and protein parameters and topologies, starting from pdb files using the standard GROMACS pdb2gmx method. CMAP is currently not implemented.

Parametrization and Application of a Coarse Grained Force Field for Benzene/Fullerene Interactions with Lipids

Recently, we reported new coarse grain (CG) force fields for lipids and phenyl/fullerene based molecules. Here, we developed the cross parameters necessary to unite those force fields and then applied the model to investigate the nature of benzene and C(60) interactions with lipid bilayers. The interaction parameters between the phenyl and lipid CG sites are based on experimental and all atom (AA) molecular dynamics (MD) data. The resulting force field was tested on benzene rich lipid bilayers and shown to reproduce general behavior expected from experiments. The parameters were then applied to C(60) interactions with lipid bilayers. Overall, the results showed excellent agreement with AA MD and experimental observations. In the C(60) lipid systems, the fullerenes were shown to aggregate even at the lowest concentrations investigated.

Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types.

A significant modification to the additive all-atom CHARMM lipid force field (FF) is developed and applied to phospholipid bilayers with both choline and ethanolamine containing head groups and with both saturated and unsaturated aliphatic chains. Motivated by the current CHARMM lipid FF (C27 and C27r) systematically yielding values of the surface area per lipid that are smaller than experimental estimates and gel-like structures of bilayers well above the gel transition temperature, selected torsional, Lennard-Jones and partial atomic charge parameters were modified by targeting both quantum mechanical (QM) and experimental data. QM calculations ranging from high-level ab initio calculations on small molecules to semiempirical QM studies on a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer in combination with experimental thermodynamic data were used as target data for parameter optimization. These changes were tested with simulations of pure bilayers at high hydration of the following six lipids: DPPC, 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE); simulations of a low hydration DOPC bilayer were also performed. Agreement with experimental surface area is on average within 2%, and the density profiles agree well with neutron and X-ray diffraction experiments. NMR deuterium order parameters (S(CD)) are well predicted with the new FF, including proper splitting of the S(CD) for the aliphatic carbon adjacent to the carbonyl for DPPC, POPE, and POPC bilayers. The area compressibility modulus and frequency dependence of (13)C NMR relaxation rates of DPPC and the water distribution of low hydration DOPC bilayers also agree well with experiment. Accordingly, the presented lipid FF, referred to as C36, allows for molecular dynamics simulations to be run in the tensionless ensemble (NPT), and is anticipated to be of utility for simulations of pure lipid systems as well as heterogeneous systems including membrane proteins.