CHARMM-GUI Membrane Builder Toward Realistic Biological Membrane Simulations

This is the peer reviewed version of the following article: Wu, E. L., Cheng, X., Jo, S., Rui, H., Song, K. C., Dávila-Contreras, E. M., … Im, W. (2014). CHARMM-GUI Membrane Builder Toward Realistic Biological Membrane Simulations. Journal of Computational Chemistry, 35(27), 1997–2004. http://doi.org/10.1002/jcc.23702, which has been published in final form at http://doi.org/10.1002/jcc.23702. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.CHARMM-GUI Membrane Builder, http://www.charmm-gui.org/input/membrane, is a web-based user interface designed to interactively build all-atom protein/membrane or membrane-only systems for molecular dynamics simulation through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builderthat allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types such as phosphoinositides, cardiolipin, sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance-based algorithm for faster initial ion displacement, (5) CHARMM inputs for P21 image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: cardiolipin lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM-GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments

NMR-Based Computational Studies of Membrane Proteins in Explicit Membranes

Since nuclear magnetic resonance (NMR) spectroscopy data, including solution NMR from micelles and solid-state NMR from bilayers, provide valuable structural and dynamics information of membrane proteins, they are commonly used as restraints in structural determination methods for membrane proteins. However, most of these methods determine the protein structures by fitting the single-confer model into all available NMR restraints regardless of the explicit environmental effects that are determinant in the structures of membrane proteins. To develop a reliable protocol for obtaining optimal structures of membrane proteins in their native-like environments, various NMR properties were applied in the refinement approaches using explicit molecular dynamics (MD) simulations in this research. First, solution NMR NOE based-distance measurements were used as restraints in MD simulations to refine an activating immunoreceptor complex in explicit environments. Compared to the structure determined in vacuum, the resulting structures from the explicit restrained simulations yields a more favorable and realistic side-chain arrangement of a key Asp residue, which is highly consistent with mutagenesis studies on such residue. Incorporating solid-state NMR and solution NMR, MD simulations were performed in the explicit bilayers to refine the structure of membrane-bound Pf1 coat protein. Since solid-state NMR is sparse in its N-terminal periplasmic helix, the protein structure was determined by combining solid-state NMR and solution NMR. Benefiting from the sophisticated energy function and the explicit environments in MD, the orientation of Pf1's periplasmic helix can be identified in simulations restrained by solid-state NMR alone. In the simulations restrained with both solid-state NMR and solution NMR, physically irrelevant structures were frequently observed, suggesting there are conflicts between the restraints from different sample types (e.g., bilayers and micelles). As NMR data are ensemble-averaged measures, the solid-state NMR restrained explicit ensemble dynamics (ED) simulations of fd coat protein were performed in different ensemble sizes and compared to the unrestrained MD simulations. As the ensemble size increases, the violations of resulting structures from experimental NMR data decrease, while the structural variations increase to be comparable to the unrestrained MD simulations, indicating the efficacy of restrained ED in refining structures and extracting dynamics. To investigate the influence of different environments on the structures of membrane proteins, in this research, MD simulations were performed in bilayers and micelles, respectively. Since building a preassembled protein/micelle complex for MD simulation is challenging and requires considerable experience with simulation software, a web-based graphical interface Micelle Builder in CHARMM-GUI (http://www.charmm-gui.org/input/micelle) was developed to support users to build micelle systems in a automatic and simplified process. Using this interface, Pf1 coat protein was preassembled in a protein/micelle model and simulated in explicit environment. Compared to previous simulations of Pf1 coat protein in bilayers, different protein conformations were observed in these simulations due to the distinct behavior and geometry of micelles

Boron Uptake in Salt Cedars via Aquaporins

Salt Cedar (Tamarix) is a dicot plant highly tolerant to the chemical boron. This is interesting because for most plants boron is an essential yet toxic metalloid. Plants have a hard time excluding it. The goal of the project is to identify a potential protein sequence (order of amino acids forming a protein) for an aquaporin that allows the transport of boron, moving through a pore. In addition to selecting the sequences, a 3D model of the protein has been constructed to see how boron is entering the cells through the channels of these proteins. A dynamic model is being made to examine the structure in a cell membrane. We have assembled 3D models of these channel proteins using computer software programs that build models based on the sequences. The sequence of a protein determines how it works. Changing the sequence changes how it works. Dynamic modeling the protein’s structure has begun, to see how the structure fluctuates. The diameter of the channel/pore is a critical value being calculated. In the static model the pore was not large enough for boron to pass through. In the dynamic model the pore should have a larger size at times. The pore size will determine if boron will fit through the channel. We expect this channel to have a lower presence in the roots of this plant, thus limiting boron uptake. This research is important because plants are currently facing boron tolerance issues across the world and particularly in the southwest region in the United States

Bioinformatics for Membrane Lipid Simulations: Models, Computational Methods, and Web Server Tools

Biological membranes are complex environments consisting of different types of lipids and membrane proteins. The structure of a lipid bilayer is typically difficult to study because the membrane liquid crystalline state is made up of multiple disordered lipid molecules. This complicates the description of the lipid membrane properties by the conformation of any single lipid molecule. Molecular dynamics (MD) simulations have been used extensively to investigate properties of membrane lipids, lipid vesicles, and membrane protein systems. All-atom membrane models can elucidate detailed contacts between membrane proteins and its surrounding lipids, while united-atom and coarse-grained description have allowed larger models and longer timescales up to microsecond mark to be probed. Additionally, membrane models with mixed phospholipids and lipopolysaccharide content have made it possible to model improved views of biological membranes. Here, we present an overview of commonly used lipid force fields by the biosimulation community, useful tools for membrane MD simulations, and recent advances in membrane simulations

A Continuum Poisson-Boltzmann Model for Membrane Channel Proteins

Membrane proteins constitute a large portion of the human proteome and perform a variety of important functions as membrane receptors, transport proteins, enzymes, signaling proteins, and more. The computational studies of membrane proteins are usually much more complicated than those of globular proteins. Here we propose a new continuum model for Poisson-Boltzmann calculations of membrane channel proteins. Major improvements over the existing continuum slab model are as follows: 1) The location and thickness of the slab model are fine-tuned based on explicit-solvent MD simulations. 2) The highly different accessibility in the membrane and water regions are addressed with a two-step, two-probe grid labeling procedure, and 3) The water pores/channels are automatically identified. The new continuum membrane model is optimized (by adjusting the membrane probe, as well as the slab thickness and center) to best reproduce the distributions of buried water molecules in the membrane region as sampled in explicit water simulations. Our optimization also shows that the widely adopted water probe of 1.4 {\AA} for globular proteins is a very reasonable default value for membrane protein simulations. It gives an overall minimum number of inconsistencies between the continuum and explicit representations of water distributions in membrane channel proteins, at least in the water accessible pore/channel regions that we focus on. Finally, we validate the new membrane model by carrying out binding affinity calculations for a potassium channel, and we observe a good agreement with experiment results.Comment: 40 pages, 6 figures, 5 table

Substrate-Specific Inhibition Constants for Phospholipase A2 Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics.

Assaying lipolytic enzymes is extremely challenging because they act on water-insoluble lipid substrates, which are normally components of micelles, vesicles, and cellular membranes. We extended a new lipidomics-based liquid chromatographic-mass spectrometric assay for phospholipases A2 to perform inhibition analysis using a variety of commercially available synthetic and natural phospholipids as substrates. Potent and selective inhibitors of three recombinant human enzymes, including cytosolic, calcium-independent, and secreted phospholipases A2 were used to establish and validate this assay. This is a novel use of dose-response curves with a mixture of phospholipid substrates, not previously feasible using traditional radioactive assays. The new application of lipidomics to developing assays for lipolytic enzymes revolutionizes in vitro testing for the discovery of potent and selective inhibitors using mixtures of membranelike substrates

A machine learning assessment of the two states model for lipid bilayer phase transitions

We have adapted a set of classification algorithms, also known as Machine Learning, to the identification of fluid and gel domains close to the main transition of dipalmitoyl-phosphatidylcholine (DPPC) bilayers. Using atomistic molecular dynamics conformations in the low and high temperature phases as learning sets, the algorithm was trained to categorize individual lipid configurations as fluid or gel, in relation with the usual two-states phenomenological description of the lipid melting transition. We demonstrate that our machine can learn and sort lipids according to their most likely state without prior assumption regarding the nature of the order parameter of the transition. Results from our machine learning approach provides strong support in favor of a two-states model approach of membrane fluidity

Orientation of Fluorescent Lipid Analog BODIPY-PC to Probe Lipid Membrane Properties: Insights from Molecular Dynamics Simulations

Single-molecule fluorescence measurements have been used to characterize membrane properties, and recently showed a linear evolution of the fluorescent lipid analog BODIPY-PC towards small tilt angles in Langmuir-Blodgett monolayers as the lateral surface pressure is increased. In this work, we have performed comparative molecular dynamics (MD) simulations of BODIPY-PC in DPPC (dipalmitoylphosphatidylcholine) monolayers and bilayers at three surface pressures (3, 10, and 40 mN/m) to explore 1) the microscopic correspondence between monolayer and bilayer structures, 2) the fluorophore’s position within the membrane, and 3) the microscopic driving forces governing the fluorophore’s tilting. The MD simulations reveal very close agreement between the monolayer and bilayer systems in terms of the fluorophore’s orientation and lipid chain order, suggesting that monolayer experiments can be used to approximate bilayer systems. The simulations capture the trend of reduced tilt angle of the fluorophore with increasing surface pressure as seen in the experimental results, and provide detailed insights into fluorophore location and orientation, not obtainable in the experiments. The simulations also reveal that the enthalpic contribution is dominant at 40 mN/m resulting in smaller tilt angles of the fluorophore, and the entropy contribution is dominant at lower pressures resulting in larger tilt angles

On the structure and stability of novel cationic DPPC liposomes doped with gemini surfactants

A novel formulation of cationic liposomes was studied by mixing dipalmitoylphosphatidylcholine (DPPC) with tetradecyltrimethylammonium bromide gemini surfactants with different alkane spacer groups lengths attached to their ammonium head-groups. The physicochemical characterization of the cationic liposomes was obtained by combining experimental results from differential scanning microcalorimetry (DSC) with molecular dynamic simulations, in order to understand their structural configuration. An adapted Ising model was used to interpret the results in terms of cooperativity of the phase transitions. The gemini surfactants partition into the lipid bilayer of DPPC liposomes, and the induced changes in colloidal stability and phase transition were analyzed in detail. The DPPC liposomes became positively charged upon gemini surfactant partition, showing increased colloidal stability. Our results show significant differences in structural configuration between gemini surfactants with short and long spacer lengths. While gemini with shorter spacers allocate within the lipid bilayer with both headgroups in the same layer, geminis with longer spacers unexpectedly intercalate in the lipid membrane in a particular zig-zag configuration, with each headgroup located at a different side of the bilayer, altering the coupling degree parameters of the membrane’s phase transition. The extraordinary increase of colloidal stability of DPPC liposomes with gemini surfactants at very low molar ratio and the possibility to tune the physicochemical properties of the membrane by control de spacer length of the geminis opens new possibilities for cationic liposomal formulations with potential applications in vaccines, drug/gene delivery or biosensingThis work was supported by the Spanish Research Agency (AEI) under Project PID2019-109517RB-I00. ERDF funds are also acknowledged. Facilities provided by the Galician Supercomputing Centre (CESGA) are also acknowledgedS