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EmrE is a bacterial efflux protein in the small multidrug-resistant (SMR) family present in Escherichia coli . Due to its small size, 110 residues in each dimer subunit, it is an ideal model system to study ligand-protein-membrane interactions. Here in our work, we have calculated the free energy landscape of benzyltrimetylammonium (BTMA) and tetraphenyl phosphonium (TPP) binding to EmrE using the enhanced sampling method-multiple walker metadynamics. We estimate that the free energy of BTMA binding to EmrE is -21.2 ± 3.3 kJ/mol and for TPP is -43.6 ± 3.8 kJ/mol. BTMA passes through two metastable states to reach the binding pocket, while TPP has a more complex binding landscape with four metastable states and one main binding site. Our simulations show that the ligands interact with the membrane lipids at a distance 1 nm away from the binding site which forms a broad local minimum, consistent for both BTMA and TPP. This site can be an alternate entry point for ligands to partition from the membrane into the protein, especially for bulky and/or branched ligands. We also observed the membrane lipid and C-terminal 110HisA form salt-bridge interactions with the helix-1 residue 22LysB. Our free energy estimates and clusters are in close agreement with experimental data and give us an atomistic view of the ligand-protein-lipid interactions. Understanding the binding pathway of these ligands can guide us in future design of ligands that can alter or halt the function of EmrE.

Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.

Membrane contacts sites (MCSs) play important roles in lipid trafficking across cellular compartments and maintain the widespread structural diversity of organelles. We have utilized microsecond long all-atom (AA) molecular dynamics (MD) simulations and enhanced sampling techniques to unravel the MCS structure targeting by yeast oxysterol binding protein (Osh4) in an environment that mimics the interface of membranes with an increased proportion of anionic lipids using CHARMM36m forcefield with additional CUFIX parameters for lipid-protein electrostatic interactions. In a dual-membrane environment, unbiased MD simulations show that Osh4 briefly interacts with both membranes, before aligning itself with a single membrane, adopting a β-crease-bound conformation similar to observations in a single-membrane scenario. Targeted molecular dynamics simulations followed by microsecond-long AA MD simulations have revealed a distinctive dual-membrane bound state of Osh4 at MCS, wherein the protein interacts with the lower membrane via the β-crease surface, featuring its PHE-239 residue positioned below the phosphate plane of membrane, while concurrently establishing contact with the opposite membrane through the extended α6-α7 region. Osh4 maintains these dual membrane contacts simultaneously over the course of microsecond-long MD simulations. Moreover, binding energy calculations highlighted the essential roles played by the phenylalanine loop and the α6 helix in dynamically stabilizing dual-membrane bound state of Osh4 at MCS. Our computational findings were corroborated through frequency of contact analysis, showcasing excellent agreement with past experimental cross-linking data. Our computational study reveals a dual-membrane bound conformation of Osh4, providing insights into protein-membrane interactions at membrane contact sites and their relevance to lipid transfer processes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jeffery Klauda reports financial support was provided by University of Maryland. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The inclusion of accurate yet computationally inexpensive lipid force fields (FF) is pertinent for the study of lipids and lipid-containing systems using molecular dynamics (MD). Within the past decade, the implementation and further expansion of a united atom (UA) FF for lipids have been developed in the CHARMM family of FFs. The most recent version of the UA presented more accurate descriptions of lipid properties for several phospholipids with saturated and monounsaturated chains, termed C36UAr. However, the original C36UAr model lacks parameters for an important class of lipids, such as sphingolipids. The focus of this article is to broaden the scope of the C36UAr chain model to incorporate these lipids. In this study, two common sphingolipids, N -palmitoyl sphingomyelin and N -stearoyl sphingomyelin are converted to a UA-chain representation and simulated to investigate the accuracy and speed over the all-atom FF model for sphingolipids. Improvements were found among multiple parameters, for example, in the surface area per lipid (SA/lip) and hydrogen order parameters, over the all-atom simulations of these sphingomyelins in C36, while as much as halving the simulation time for simulations of the same setup otherwise. Thus, the accuracy and efficiency found in this study are consistent with those found in the C36UAr model for phospholipids and expand the application of C36UAr to a wider array of membrane models to better match that in vivo.

The SARS-CoV-2 ORF7b protein has drawn attention for its potential role in viral pathogenesis, but its structural details and lateral membrane associations remain elusive. In this study, we conducted multiscale molecular dynamics simulations to provide detailed molecular insights of the protein's dimerization, which is crucial for unraveling its structural model of protein-protein interface important to regulating cellular immune response. To gain a deeper understanding of homodimer configurations, we employed a machine learning algorithm for structural-based clustering. Clusters were categorized into three distinct groups for both parallel and antiparallel orientations, highlighting the influence of the initial monomer conformation on dimer configurations. Analysis of hydrogen bonding and π-π and π-cation stacking interactions within clusters revealed variations in interactions between clusters. In parallel dimers, weak stacking interactions in the transmembrane (TM) region were observed. In contrast, antiparallel dimers exhibited strong hydrogen bonding and stacking interactions contributing to tight dimeric packing, both within and outside the TM domain. Overall, our study provides a comprehensive view of the structural dynamics of ORF7b homodimerization in both parallel and antiparallel orientations. These findings shed light on the molecular interactions involved in ORF7b dimerization, which are crucial for understanding its potential roles in SARS-CoV-2 pathogenesis. This knowledge could inform future research and therapeutic strategies targeting this viral protein.

Molecular dynamics (MD) simulations are a useful tool when studying the properties of membranes as they allow for a molecular view of lipid interactions with proteins, nucleic acids, or small molecules. While model membranes are usually symmetric in their lipid composition between leaflets and include a small number of lipid components, physiological membranes are highly complex and vary in the level of asymmetry. Simulation studies have shown that changes in leaflet asymmetry can alter the properties of a membrane. It is therefore necessary to carefully build asymmetric membranes to accurately simulate membranes. This chapter carefully describes the different methods for building asymmetric membranes and the advantages/disadvantages of each method. The simplest methods involve building a membrane with either an equal number of lipids per leaflet or an equal initial surface area (SA) estimated by the area per lipid. More detailed methods include combining two symmetric membranes of equal SA or altering an asymmetric membrane and adjusting the number of lipids after equilibration to minimize an observable such as differential stress (0-DS). More complex methods that require specific simulation software are also briefly described. The challenges and assumptions are listed for each method which should help guide the researcher to choose the best method for their unique MD simulation of an asymmetric membrane., (Copyright © 2024. Published by Elsevier Inc.)

Macrophages (MAs), which play vital roles in human immune responses and lipid metabolisms, are implicated in the development and progression of atherosclerosis, a major contributor to cardiovascular diseases. Specifically, the abnormal lipid metabolism of oxidized low-density lipids (oxLDLs) in MAs is believed to be a crucial factor. However, the precise mechanism by which the MA membrane contributes to this altered lipid metabolism remains unclear. Lipidomic studies have revealed significant differences in membrane composition between various MA phenotypes. This study serves to provide and characterize complex realistic computational models for naïve (M0) and Kdo2-lipid A-activated (M1) state MA. Analyses of surface area per lipid (SA/lip), area compressibility modulus (K A ), carbon‑hydrogen order parameter (S CH ), electron density profile (EDP), tilt angles, two-dimension radial distribution functions (2D RDFs), mean squared displacement (MSD), hydrogen bonds (H-bonds), lipid clustering, and lipid wobble were conducted for both models. Results indicate that the M1 state MA membrane is more tightly packed, with increased chain order across lipid species, and forms PSM-DOPG-CHOL and PSM-SLPC-CHOL clusters. Importantly, the bilayer thicknesses reported for the models are in good agreement with experimental data for the thicknesses of transmembrane regions for MA integral proteins. These findings validate the described models as physiologically accurate for future computational studies of MA membranes and their residing proteins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jeffery Klauda reports financial support was provided by National Science Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Conformational dynamics in proteins can give rise to aggregation prone states during folding, and these kinetically stable states could form oligomers and aggregates. In this study, we investigate the intermediate states and near-folded states of β2-microglobulin and their physico-chemical properties using molecular dynamics and Markov state modeling. Analysis of hundreds of microseconds simulation show the importance of the edge strands in the misfolded states that give rise to a high exposure of hydrophobic residues in the core of the protein that could initiate oligomerization and aggregate formation. Our study sheds light on the first step of aggregation of β2m monomers and gave a better picture of the landscape of protein misfolding and aggregation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bryan Berger and Jeff Klauda reports financial support was provided by National Science Foundation. The co-author Jeff Klauda serves on the editorial board of BBA Biomembranes. Bryan Berger and Jeff Klauda reports financial support was provided by National Science Foundation. The co-author Jeff Klauda serves on the editorial board of BBA Biomembranes., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Accurate empirical force fields of lipid molecules are a critical component of molecular dynamics simulation studies aimed at investigating properties of monolayers, bilayers, micelles, vesicles, and liposomes, as well as heterogeneous systems, such as protein-membrane complexes, bacterial cell walls, and more. While the majority of lipid force field-based simulations have been performed using pairwise-additive nonpolarizable models, advances have been made in the development of the polarizable force field based on the classical Drude oscillator model. In the present study, we undertake further optimization of the Drude lipid force field, termed Drude2023, including improved treatment of the phosphate and glycerol linker region of PC and PE headgroups, additional optimization of the alkene group in monounsaturated lipids, and inclusion of long-range Lennard-Jones interactions using the particle-mesh Ewald method. Initial optimization targeted quantum mechanical (QM) data on small model compounds representative of the linker region. Subsequent optimization targeted QM data on larger model compounds, experimental data, and dihedral potentials of mean force from the CHARMM36 additive lipid force field using a parameter reweighting protocol. The use of both experimental and QM target data during the reweighting protocol is shown to produce physically reasonable parameters that reproduce a collection of experimental observables. Target data for optimization included surface area/lipid for DPPC, DSPC, DMPC, and DLPC bilayers and nuclear magnetic resonance (NMR) order parameters for DPPC bilayers. Validation data include prediction of membrane thickness, scattering form factors, electrostatic potential profiles, compressibility moduli, surface area per lipid, water permeability, NMR T 1 relaxation times, diffusion constants, and monolayer surface tensions for a variety of saturated and unsaturated lipid mono- and bilayers. Overall, the agreement with experimental data is quite good, though the results are less satisfactory for the NMR T 1 relaxation times for carbons near the ester groups. Notable improvements compared to the additive C36 force field were obtained for membrane dipole potentials, lipid diffusion coefficients, and water permeability with the exception of monounsaturated lipid bilayers. It is anticipated that the optimized polarizable Drude2023 force field will help generate more accurate molecular simulations of pure bilayers and heterogeneous systems containing membranes, advancing our understanding of the role of electronic polarization in these systems.

The human ocular lens consists primarily of elongated, static fibers characterized by high stability and low turnover, which differ dramatically in their composition and properties from other biological membranes. Cholesterol (Chol) and sphingolipids (SL) are present at high concentrations, including saturated SLs, such as dihyrosphingomyelin (DHSM). Past molecular dynamics simulations demonstrated that the presence of DHSM and high Chol concentration contributes to higher order in lipid membranes. This current study simulated more complex models of human lens membranes. Models were developed representing physiological compositions in cataractous lenses aged 74 ± 6 years and in healthy lenses aged 22 ± 4, 41 ± 6, and 69 ± 3 years. With older age, Chol and ceramide concentrations increase and glycerophospholipid concentration decreases. With cataract, ceramide concentration increases and Chol and glycerophospholipid concentrations decrease. Surface area per lipid, deuterium order parameters (S CD ), sterol tilt angle, electron density profiles, bilayer thickness, chain interdigitation, two-dimensional radial distribution functions (2D-RDF), lipid clustering, and hydrogen bonding were calculated for all simulations. All systems exhibited low surface area per lipid and high bilayer thickness, indicative of strong vertical packing. S CD parameters suggest similarly, with saturated tails in the hydrophobic core of the membrane having elevated order. Vertical packing and acyl tail order increased with both age and cataract condition. Lateral diffusion decreased with age and cataracts, with the older and cataractous models demonstrating increased long-range structure by the 2D-RDF analysis. In future work examining the membrane proteins of the lens, these models can serve as a physiologically accurate representation of the lens lipidome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In an effort to combat rising antimicrobial resistance, our labs have rationally designed cationic, helical, amphipathic antimicrobial peptides (AMPs) as alternatives to traditional antibiotics since AMPs incur bacterial resistance in weeks, rather than days. One highly positively charged AMP, WLBU2 (+13 e ), ( RR WV RR V R R WV R R VV R VV RR WV RR ), has been shown to be effective in killing both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria by directly perturbing the bacterial membrane nonspecifically. Previously, we used two equilibrium experimental methods: synchrotron X-ray diffuse scattering (XDS) providing lipid membrane thickness and neutron reflectometry (NR) providing WLBU2 depth of penetration into three lipid model membranes (LMMs). The purpose of the present study is to use the results from the scattering experiments to guide molecular dynamics (MD) simulations to investigate the detailed biophysics of the interactions of WLBU2 with LMMs of Gram-negative outer and inner membranes, and Gram-positive cell membranes, to elucidate the mechanisms of bacterial killing. Instead of coarse-graining, backmapping, or simulating without bias for several microseconds, all-atom (AA) simulations were guided by the experimental results and then equilibrated for ∼0.5 μs. Multiple replicas of the inserted peptide were run to probe stability and reach a combined time of at least 1.2 μs for G(-) and also 2.0 μs for G(+). The simulations with experimental comparisons help rule out certain structures and orientations and propose the most likely set of structures, orientations, and effects on the membrane. The simulations revealed that water, phosphates, and ions enter the hydrocarbon core when WLBU2 is positioned there. For an inserted peptide, the three types of amino acids, arginine, tryptophan, and valine (R, W, V), are arranged with the 13 Rs extending from the hydrocarbon core to the phosphate group, Ws are located at the interface, and Vs are more centrally located. For a surface state, R, W, and V are positioned relative to the bilayer interface as expected from their hydrophobicities, with Rs closest to the phosphate group, Ws close to the interface, and Vs in between. G(-) and G(+) LMMs are thinned ∼1 Å by the addition of WLBU2. Our results suggest a dual anchoring mechanism for WLBU2 both in the headgroup and in the hydrocarbon region that promotes a defect region where water and ions can flow across the slightly thinned bacterial cell membrane.

The cyclin-dependent kinase (CDK5) forms a stable complex with its activator p25, leading to the hyperphosphorylation of tau proteins and to the formation of plaques and tangles that are considered to be one of the typical causes of Alzheimer's disease (AD). Hence, the pathological CDK5-p25 complex is a promising therapeutic target for AD. Small peptides, obtained from the truncation of CDK5 physiological activator p35, have shown promise in inhibiting the pathological complex effectively while also crossing the blood-brain barrier. One such small 24-residue peptide, p5, has shown selective inhibition toward the pathological complex in vivo. Our previous research focused on the characterization of a computationally predicted CDK5-p5 binding mode and of its pharmacophore, which was consistent with competitive inhibition. In continuation of our previous work, herein, we investigate four additional binding modes to explore other possible mechanisms of interaction between CDK5 and p5. The quantitative description of the pharmacophore is consistent with both competitive and allosteric p5-induced inhibition mechanisms of CDK5-p25 pathology. The gained insights can direct further in vivo/in vitro tests and help design small peptides, linear or cyclic, or peptidomimetic compounds as adjuvants of orthosteric inhibitors or as part of a cocktail of drugs with enhanced effectiveness and lower side effects.

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3-mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell-based immunotherapy.

Finding a low dimensional representation of data from long-timescale trajectories of biomolecular processes, such as protein folding or ligand-receptor binding, is of fundamental importance, and kinetic models, such as Markov modeling, have proven useful in describing the kinetics of these systems. Recently, an unsupervised machine learning technique called VAMPNet was introduced to learn the low dimensional representation and the linear dynamical model in an end-to-end manner. VAMPNet is based on the variational approach for Markov processes and relies on neural networks to learn the coarse-grained dynamics. In this paper, we combine VAMPNet and graph neural networks to generate an end-to-end framework to efficiently learn high-level dynamics and metastable states from the long-timescale molecular dynamics trajectories. This method bears the advantages of graph representation learning and uses graph message passing operations to generate an embedding for each datapoint, which is used in the VAMPNet to generate a coarse-grained dynamical model. This type of molecular representation results in a higher resolution and a more interpretable Markov model than the standard VAMPNet, enabling a more detailed kinetic study of the biomolecular processes. Our GraphVAMPNet approach is also enhanced with an attention mechanism to find the important residues for classification into different metastable states.

All-atom (AA) molecular dynamics simulations are used to unravel the binding mechanism of yeast oxysterol binding protein (Osh4) to model membranes with varying anionic lipid concentration using AA and the highly mobile membrane mimetic (HMMM) representations. For certain protein-lipid interactions, an improved forcefield description is used (CUFIX) to accurately describe lipid-protein electrostatic interactions. Our detailed computational studies have identified a single, β-crease oriented, membrane-bound conformation of Osh4 for all anionic membranes. The penetration of the PHE-239 residue below the membrane phosphate plane is the characteristic signature of the membrane-bound state of Osh4. As the phenylalanine loop anchors itself deeply in the membrane; the other regions of the Osh4, namely, ALPS motif, β6- β7 loop, β14- β15 loop, and β16- β17 loop, maximize their contact with the membrane. Furthermore, loose lipid packing and higher mobility of HMMM enable stronger association of the ALPS motif with the membrane lipids through its hydrophobic surface. After the HMMM is converted to AA and equilibrated, the binding is two to three times stronger compared with simulations started with the AA representation, yielding the major importance of the ALPS motif to binding. Quantitative estimation of binding energy revealed that the phenylalanine loop plays a crucial role in stable membrane attachment of Osh4 and contributes significantly toward overall binding process. The CUFIX parameters provide a more balanced picture of hydrophobic and electrostatic interactions between the protein and the membrane, which differs from our past work that showed salt bridges alone stabilized Osh4-membrane contact. Our study provides a comprehensive picture of the binding mechanism of Osh4 with model single membranes and, thus, understanding of the initial interactions is important for elucidating the biological function of this protein to shuttle lipids between organelles., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Lipid composition asymmetry between leaflets is important to cell function and plays a key role in the "positive inside" rule in transmembrane proteins. In this work, Escherichia coli inner plasma membrane models reflecting this asymmetry have been investigated at the early-log and stationary stages during the bacterial lifecycle using all-atom molecular dynamics simulations. The CHARMM36 lipid force field is used, and selected membrane properties are tested for variations between two leaflets and whole membranes. Our models include bacterial lipids with a cyclopropane moiety on the sn-2 acyl chain in the stationary membrane model. The PE/PG ratio for two leaflets reflects the "positive inside" rule of membrane proteins, set to 6.8 and 2.8 for the inner and outer leaflets of the two models, respectively. We are the first to model leaflet asymmetry in the lipid composition of E. coli cytoplasmic membranes and observe the effect on membrane properties in leaflets and whole membranes. Specifically, our results show that for the stationary phase bilayer, the surface area per lipid (SA/lipid) is larger, the thickness (2 D C and D B ) is smaller, the tilt angle is larger, the tilt modulus is smaller, and the deuterium order parameters ( S CD ) of sn-1 and sn-2 tails are lower, compared to the early-log stage. Moreover, the stationary stage bilayer has a positive spontaneous curvature, while the early-log stage has a near flat spontaneous curvature. For leaflet asymmetry, the inner leaflet has a larger SA/lipid, a smaller thickness, a smaller elastic tilt modulus (a larger tilt angle), and lower S CD , compared to the outer leaflet in both stages. Moreover, an asymmetric membrane involves a lipid tilt and a lateral extension, varying from a reference state of a pre-equilibrium membrane. This work encourages a more profound exploration of leaflet asymmetry in various other membrane models and how this might affect the structure and function of membrane-associated peptides and proteins.

Cell membranes are composed of a variety of lipids and proteins where they interact with each other to fulfill their roles. The first step in modeling these interactions in molecular simulations is to have reliable mimetics of the membrane's lipid environment. This Feature Article presents our recent efforts to model complex cellular membranes using all-atom force fields. A short review of the CHARMM36 (C36) lipid force field and its recent update to incorporate the long-range dispersion is presented. Key examples of model membranes mimicking various species and organelles are given. These include single-celled organisms such as bacteria ( E. coli. , chlamydia, and P. aeruginosa ) and yeast (plasma membrane, endoplasmic reticulum, and trans-Golgi network) and more advanced ones such as plants (soybean and Arabidopsis thaliana ) and mammals (ocular lens, stratum corneum, and peripheral nerve myelin). Leaflet asymmetry in composition has also been applied to some of these models. With the increased lipid diversity in the C36 lipid FF, these complex models can better reflect the structural, mechanical, and dynamic properties of realistic membranes and open an opportunity to study biological processes involving other molecules.

Conformational sampling of biomolecules using molecular dynamics simulations often produces a large amount of high dimensional data that makes it difficult to interpret using conventional analysis techniques. Dimensionality reduction methods are thus required to extract useful and relevant information. Here, we devise a machine learning method, Gaussian mixture variational autoencoder (GMVAE), that can simultaneously perform dimensionality reduction and clustering of biomolecular conformations in an unsupervised way. We show that GMVAE can learn a reduced representation of the free energy landscape of protein folding with highly separated clusters that correspond to the metastable states during folding. Since GMVAE uses a mixture of Gaussians as its prior, it can directly acknowledge the multi-basin nature of the protein folding free energy landscape. To make the model end-to-end differentiable, we use a Gumbel-softmax distribution. We test the model on three long-timescale protein folding trajectories and show that GMVAE embedding resembles the folding funnel with folded states down the funnel and unfolded states outside the funnel path. Additionally, we show that the latent space of GMVAE can be used for kinetic analysis and Markov state models built on this embedding produce folding and unfolding timescales that are in close agreement with other rigorous dynamical embeddings such as time independent component analysis.

Arabidopsis thaliana is an important model organism, which has attracted many biologists. While most research efforts have been on studying the genetics and proteins of this organism, a systematic study of its lipidomics is lacking. Here, we present a novel, asymmetric model of its cell membrane with its lipid composition consisting of five glycerophospholipids, two glycolipids, and sitosterol determined from multiple independent experiments. A typical lipid type in plant membranes is glycosyl inositol phosphoryl ceramide (GIPC), which accounts for about 10% of the total lipids in the outer leaflet in our model. Two symmetric models representing the inner and outer leaflets of the membrane were built and simulated until equilibrium was reached and then combined to form the asymmetric model. Our results indicate that the outer leaflet is more rigid and tightly packed compared to the inner leaflet. Pressure profiles for the two leaflets are overall similar though the outer leaflet exhibits larger oscillations. A special focus on lipid organization is discussed and the interplay between glycolipids and sitosterols is found to be important. The current model provides a baseline for future modeling of similar membranes and can be used to study partitioning of small molecules in the membrane or further developed to study the interaction between plant membrane proteins and lipids.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 continues to rage with devastating consequences on human health and global economy. The spike glycoprotein on the surface of coronavirus mediates its entry into host cells and is the target of all current antibody design efforts to neutralize the virus. The glycan shield of the spike helps the virus to evade the human immune response by providing a thick sugar-coated barrier against any antibody. To study the dynamic motion of glycans in the spike protein, we performed microsecond-long molecular dynamics simulation in two different states that correspond to the receptor binding domain in open or closed conformations. Analysis of this microsecond-long simulation revealed a scissoring motion on the N-terminal domain of neighboring monomers in the spike trimer. The roles of multiple glycans in shielding of spike protein in different regions were uncovered by a network analysis, in which the high betweenness centrality of glycans at the apex revealed their importance and function in the glycan shield. Microdomains of glycans were identified featuring a high degree of intracommunication in these microdomains. An antibody overlap analysis revealed the glycan microdomains as well as individual glycans that inhibit access to the antibody epitopes on the spike protein. Overall, the results of this study provide detailed understanding of the spike glycan shield, which may be utilized for therapeutic efforts against this crisis., (Copyright © 2021 Biophysical Society. All rights reserved.)

The multilayered myelin sheath is a critical component of both central and peripheral nervous systems, forming a protective barrier against axonal damage and facilitating the movement of nervous impulses. It is primarily composed of cholesterol (CHL1), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM), and galactosylceramide (GalCer) lipids. For rat sciatic nerve myelin (part of the peripheral nervous system, PNS), it has been found that cholesterol and unsaturated fatty acid contents are significantly lower in diabetic than in non-diabetic conditions. In this study, lipid compositions from experimental data are used to create four model rat sciatic nerve myelin lipid bilayers: PI-containing (non-diabetic and diabetic) and PS-containing (non-diabetic and diabetic), which were then simulated using the all-atom CHARMM36 force field. Simulation results of diabetic membranes indicate less rigid, more laterally expansive, and thinner bilayers as well as potentially reduced interactions between GalCer on opposing myelin leaflets, supporting a direct role of the cholesterol content decrease in instigating myelin deterioration and diabetic peripheral neuropathy. Compared to PI-lipids, PS-lipids were found to cause higher inter-lipid spacing and decreased order within membranes as a result of their smaller headgroup size and higher inter-lipid hydrogen bonding potential, which allow them to more frequently reside deeper in the membrane plane and produce pushing effects on other lipids. GalCer deuterium order parameters and non-diabetic headgroup-to-headgroup bilayer thicknesses were compared to experimental data, exhibiting close alignment, supporting the future usage of these models to study the PNS myelin sheath.

Molecular simulations of biological molecules require an accurate description of molecular interactions through a force field (FF). The focus of this Perspective is on all-atom lipid FFs. Recent additions to the CHARMM36 lipid FF continue to expand a researcher's ability to probe membrane structure and function with a wide variety of biologically important lipids. Currently, there is an effort to reduce the assumptions in all-atom lipid FFs. The inclusion of long-range dispersion interaction through particle-mesh Ewald is allowing for more accurate descriptions of lipid bilayer and monolayer properties without additional computational cost. Soon, simulations with lipid FFs will no longer depend on short-range cutoffs and will accurately represent long-range dispersion. This requires efficient FF parametrization with an automated approach due to FF complexity. In addition, polarizable FFs for lipids will be important for the next generation of simulations that accurately represent how molecule interactions respond to a varied environment.

All-atom molecular dynamics simulations are used with the highly mobile membrane mimetic method to study the α 6 -α 7 peptide of the critical yeast Osh4 peripheral membrane protein. This research focuses on the impact of 1-palmitoyl-2-oleoyl- sn -glycero-phosphatidylinoside 4,5-bisphosphate (PIP2) lipids and 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-l-serine on the protein's ability to bind to the membrane. Details of the binding mechanism are described qualitatively and quantitatively by measuring the position of the deepest residues, angle of the peptide during binding, root mean square deviation of the atomic positions within the peptide, and interaction energy, while changing variables, such as the force field used and the presence of the PIP2 lipids. The negatively charged PIP2 has a large head group that is a few Ångstroms above the main membrane phosphates enabling the PIP2 lipids to interact with the peptide before it binds deeper into the membrane. The PIP2 lipids can alter the position of the peptide during binding by recruiting charged residues on the α 7 helix, such as R344 and R347. Residues R347 and R344 are unusual because they are slightly out of the reach of the main membrane phosphates but optimally positioned to interact with the PIP2 lipids. The salt-bridge interactions can also typically occur between cationic peptide residues such as R314, K325, and K336. The force field interaction effect on peptide binding was also investigated by changing the standard CHARMM36m to an improved description between some amino acids and lipid moieties ( Phys. Chem. Chem. Phys . 20 , 8432-8449). This resulted in the total number of salt bridges and hydrogen bonds being drastically reduced, the interaction energy was also reduced, and there was more balance between electrostatic and nonpolar interactions, but the general bound structure is maintained. This work is an important initial step to understand the effect of the Osh4 protein on the membrane binding and to quantify the effect of PIP2 lipids on this domain.

Halobacteria, a type of archaea in high salt environments, have phytanyl ether phospholipid membranes containing up to 50% menaquinone. It is not understood why a high concentration of menaquinone is required and how it influences membrane properties. In this study, menaquinone-8 headgroup and torsion parameters of isoprenoid tail are optimized in the CHARMM36 force field. Molecular dynamics simulations of archaeal bilayers containing 0 to 50% menaquinone characterize the distribution of menaquinone-8 and menaquinol-8, as well as their effects on mechanical properties and permeability. Menaquinone-8 segregates to the membrane midplane above concentrations of 10%, favoring an extended conformation in a fluid state. Menaquinone-8 increases the bilayer thickness but does not significantly alter the area compressibility modulus and lipid chain ordering. Counterintuitively, menaquinone-8 increases water permeability because it lowers the free energy barrier in the midplane. The thickness increase due to menaquinone-8 may help halobacteria ameliorate hyper-osmotic pressure by increasing the membrane bending constant. Simulations of the archaeal membranes with archaerhodopsin-3 show that the local membrane surface adjusts to accommodate the thick membranes. Overall, this study delineates the biophysical landscape of 50% menaquinone in the archaeal bilayer, demonstrates the mixing of menaquinone and menaquinol, and provides atomistic details about menaquinone configurations.

As a class of drugs prescribed to heart disease patients, statins are among the most popular prescription drugs in the world. Over the years, statins have been shown to have beneficial effects on patients via pathways independent of their effect on cholesterol. These pleiotropic effects vary across the different statins, and a growing hypothesis is that they are related to the localization of the statins in and their effect on the membrane. In this study, we use molecular dynamics (MD) simulations with the CHARMM36 all-atom force field to investigate the localization of statins (atorvastatin, cerivastatin, lovastatin, and pravastatin) in a POPC bilayer and how they affect the acyl chain order parameters (S CD ), surface area per lipid (APL), and thicknesses of the bilayer. The data obtained from 500 ns simulations suggests that lovastatin is localized deepest in the membrane, mostly interacting with the hydrophobic core, cerivastatin is slightly closer to the bilayer/solvent interface than lovastatin and interacts with the headgroups via its dihydroxy acid group, and pravastatin is found closest to the bilayer/solvent interface, its hydrophobic rings interacting mostly with the region around the acyl's carbonyl and its dihydroxy acid interacting with the solvent and the headgroups. Consistent binding of atorvastatin to the bilayer is not observed during our simulation due to self-aggregation. The statins differentially alter the S CD and APL and most of the bilayer thicknesses, but these effects are modest. Overall, as expected, the localization of statins seems to follow their hydrophilicity, and given previous data showing the relationship between statins' hydrophobicity and pleiotropic effects, one would expect statins that localize and interact with different regions of the membrane to have different effects. This research provides some important insight into statin localization in a simplified model of a cellular membrane., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, we report binding free energy calculations of various drugs-of-abuse to Cucurbit-[8]-uril as part of the SAMPL8 blind challenge. Force-field parameters were obtained from force-matching with different quantum mechanical levels of theory. The Replica Exchange Umbrella Sampling (REUS) approach was used with a cylindrical restraint to enhance the sampling of host-guest binding. Binding free energy was calculated by pulling the guest molecule from one side of the symmetric and cylindrical host, then into and through the host, and out the other side (bidirectional) as compared to pulling only to the bound pose inside the cylindrical host (unidirectional). The initial results with force-matched MP2 parameter set led to RMSE of 4.68 [Formula: see text] from experimental values. However, the follow-up study with CHARMM generalized force field parameters and force-matched PM6-D3H4 parameters resulted in RMSEs from experiment of [Formula: see text] and [Formula: see text], respectively, which demonstrates the potential of REUS for accurate binding free energy calculation given a more suitable description of energetics. Moreover, we compared the free energies for the so called bidirectional and unidirectional free energy approach and found that the binding free energies were highly similar. However, one issue in the bidirectional approach is the asymmetry of profile on the two sides of the host. This is mainly due to the insufficient sampling for these larger systems and can be avoided by longer sampling simulations. Overall REUS shows great promise for binding free energy calculations.

The development of the CHARMM lipid force field (FF) can be traced back to the early 1990s with its current version denoted CHARMM36 (C36). The parametrization of C36 utilized high-level quantum mechanical data and free energy calculations of model compounds before parameters were manually adjusted to yield agreement with experimental properties of lipid bilayers. While such manual fine-tuning of FF parameters is based on intuition and trial-and-error, automated methods can identify beneficial modifications of the parameters via their sensitivities and thereby guide the optimization process. This work introduces a semi-automated approach to reparametrize the CHARMM lipid FF with consistent inclusion of long-range dispersion through the Lennard-Jones particle-mesh Ewald (LJ-PME) approach. The optimization method is based on thermodynamic reweighting with regularization with respect to the C36 set. Two independent optimizations with different topology restrictions are presented. Targets of the optimizations are primarily liquid crystalline phase properties of lipid bilayers and the compression isotherm of monolayers. Pair correlation functions between water and lipid functional groups in aqueous solution are also included to address headgroup hydration. While the physics of the reweighting strategy itself is well-understood, applying it to heterogeneous, complex anisotropic systems poses additional challenges. These were overcome through careful selection of target properties and reweighting settings allowing for the successful incorporation of the explicit treatment of long-range dispersion, and we denote the newly optimized lipid force field as C36/LJ-PME. The current implementation of the optimization protocol will facilitate the future development of the CHARMM and related lipid force fields.

Long-range Lennard-Jones (LJ) interactions have been incorporated into the CHARMM36 (C36) lipid force field (FF) using the LJ particle-mesh Ewald (LJ-PME) method in order to remove the inconsistency of bilayer and monolayer properties arising from the exclusion of long-range dispersion [Yu, Y.; Semi-automated Optimization of the CHARMM36 Lipid Force Field to Include Explicit Treatment of Long-Range Dispersion. J. Chem. Theory Comput. 2021, 10.1021/acs.jctc.0c01326. (preceding article in this issue)]. The new FF is denoted C36/LJ-PME. While the first optimization was based on three phosphatidylcholines (PCs), this work extends the validation and parametrization to more lipids including PC, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and ether lipids. The agreement with experimental structure data is excellent for PC, PE, and ether lipids. C36/LJ-PME also compares favorably with scattering data of PG bilayers but less so with NMR deuterium order parameters of 1,2-dimyristoyl- sn -glycero-3-phospho-(1'-rac-glycerol) (DMPG) at 303.15 K, indicating a need for future optimization regarding PG-specific parameters. Frequency dependence of NMR T 1 spin-lattice relaxation times is well-described by C36/LJ-PME, and the overall agreement with experiment is comparable to C36. Lipid diffusion is slower than C36 due to the added long-range dispersion causing a higher viscosity, although it is still too fast compared to experiment after correction for periodic boundary conditions. When using a 10 Å real-space cutoff, the simulation speed of C36/LJ-PME is roughly equal to C36. While more lipids will be incorporated into the FF in the future, C36/LJ-PME can be readily used for common lipids and extends the capability of the CHARMM FF by supporting monolayers and eliminating the cutoff dependence.

Hydrogen mass repartitioning (HMR) that permits time steps of all-atom molecular dynamics simulation up to 4 fs by increasing the mass of hydrogen atoms has been used in protein and phospholipid bilayers simulations to improve conformational sampling. Molecular simulation input generation via CHARMM-GUI now supports HMR for diverse simulation programs. In addition, considering ambiguous pH at the bacterial outer membrane surface, different protonation states, either -2e or -1e, of phosphate groups in lipopolysaccharides (LPS) are also supported in CHARMM-GUI LPS Modeler . To examine the robustness of HMR and the influence of protonation states of phosphate groups on LPS bilayer properties, eight different LPS-type all-atom systems with two phosphate protonation states are modeled and simulated utilizing both OpenMM 2-fs (standard) and 4-fs (HMR) schemes. Consistency in the conformational space sampled by standard and HMR simulations shows the reliability of HMR even in LPS, one of the most complex biomolecules. For systems with different protonation states, similar conformations are sampled with a PO 4 1- or PO 4 2- group, but different phosphate protonation states make slight impacts on lipid packing and conformational properties of LPS acyl chains. Systems with PO 4 1- have a slightly smaller area per lipid and thus slightly more ordered lipid A acyl chains compared to those with PO 4 2- , due to more electrostatic repulsion between PO 4 2- even with neutralizing Ca 2+ ions. HMR and different protonation states of phosphates of LPS available in CHARMM-GUI are expected to be useful for further investigations of biological systems of diverse origin.

The novel coronavirus (nCOV-2019) outbreak has put the world on edge, causing millions of cases and hundreds of thousands of deaths all around the world, as of June 2020, let alone the societal and economic impacts of the crisis. The spike protein of nCOV-2019 resides on the virion's surface mediating coronavirus entry into host cells by binding its receptor binding domain (RBD) to the host cell surface receptor protein, angiotensin converter enzyme (ACE2). Our goal is to provide a detailed structural mechanism of how nCOV-2019 recognizes and establishes contacts with ACE2 and its difference with an earlier severe acute respiratory syndrome coronavirus (SARS-COV) in 2002 via extensive molecular dynamics (MD) simulations. Numerous mutations have been identified in the RBD of nCOV-2019 strains isolated from humans in different parts of the world. In this study, we investigated the effect of these mutations as well as other Ala-scanning mutations on the stability of the RBD/ACE2 complex. It is found that most of the naturally occurring mutations to the RBD either slightly strengthen or have the same binding affinity to ACE2 as the wild-type nCOV-2019. This means that the virus had sufficient binding affinity to its receptor at the beginning of the crisis. This also has implications for any vaccine design endeavors since these mutations could act as antibody escape mutants. Furthermore, in silico Ala-scanning and long-timescale MD simulations highlight the crucial role of the residues at the interface of RBD and ACE2 that may be used as potential pharmacophores for any drug development endeavors. From an evolutional perspective, this study also identifies how the virus has evolved from its predecessor SARS-COV and how it could further evolve to become even more infectious.

Permeation of many small molecules through lipid bilayers can be directly observed in molecular dynamics simulations on the nano- and microsecond timescale. While unbiased simulations provide an unobstructed view of the permeation process, their feasibility for computing permeability coefficients depends on various factors that differ for each permeant. The present work studies three small molecules for which unbiased simulations of permeation are feasible within less than a microsecond, one hydrophobic (oxygen), one hydrophilic (water), and one amphiphilic (ethanol). Permeabilities are computed using two approaches: counting methods and a maximum-likelihood estimation for the inhomogeneous solubility diffusion (ISD) model. Counting methods yield nearly model-free estimates of the permeability for all three permeants. While the ISD-based approach is reasonable for oxygen, it lacks precision for water due to insufficient sampling and results in misleading estimates for ethanol due to invalid model assumptions. It is also demonstrated that simulations using a Langevin thermostat with collision frequencies of 1/ps and 5/ps yield oxygen permeabilities and diffusion constants that are lower than those using Nosé-Hoover by statistically significant margins. In contrast, permeabilities from trajectories generated with Nosé-Hoover and the microcanonical ensemble do not show statistically significant differences. As molecular simulations become more affordable and accurate, calculation of permeability for an expanding range of molecules will be feasible using unbiased simulations. The present work summarizes theoretical underpinnings, identifies pitfalls, and develops best practices for such simulations.

Permeation of small molecules through membranes is a fundamental biological process, and molecular dynamics simulations have proven to be a promising tool for studying the permeability of membranes by providing a precise characterization of the free energy and diffusivity. In this study, permeation of ethanol through three different membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), PO-phosphatidylethanolamine (POPE), and PO-phosphatidylcholine (POPC) is studied. Permeabilities are calculated and compared with two different approaches based on Fick's first law and the inhomogeneous solubility-diffusion model. Microsecond simulation of double bilayers of these membranes provided a direct measurement of permeability by a flux-based counting method. These simulations show that a membrane of POPC has the highest permeability, followed by POPE and POPS. Due to the membrane-modulating properties of ethanol, the permeability increases as functions of concentration and saturation of the inner leaflet in a double bilayer setting, as opposed to the customary definition as a proportionality constant. This concentration dependence is confirmed by single bilayer simulations at different ethanol concentrations ranging from 1% to 18%, where permeability estimates are available from transition-based counting and the inhomogeneous solubility-diffusion model. We show that the free energy and diffusion profiles for ethanol lack accuracy at higher permeant concentrations due to non-Markovian kinetics caused by collective behavior. In contrast, the counting method provides unbiased estimates. Finally, the permeabilities obtained from single bilayer simulations are combined to represent natural gradients felt by a cellular membrane, which accurately models the non-equilibrium effects on ethanol permeability from single bilayer simulations in equilibrium.

Accurate lipid force field (FF) parameters used in molecular dynamics (MD) simulations are crucial for understanding the properties of lipid-containing systems and biological processes related to lipids. The last update of the CHARMM36 united atom chain model (C36UA) was in 2013 [Lee, S. J. Phys. Chem. B 2014, 118, 547 556]; it utilized CHARMM36 (C36) lipid FF parameters for headgroups and OPLS-UA Lennard-Jones (LJ) parameters for tails. Simulations with the FF were able to reproduce many experimental observables of lipid bilayers accurately, but to be more applicable for a wide range of lipids, additional FF parameter optimization was needed. In this work, we present an update of the model, named C36UAr. The parameterization included the LJ parameters for hydrocarbons and related dihedrals. Bulk liquid properties (density, heat of vaporization, isothermal compressibility, and diffusion constant) of model compounds were used as targets for the LJ parameter fitting, and dihedrals were fit to either quantum mechanical (QM) or potential of mean force (PMF) calculations using C36. Thermodynamic reweighting was used to further improve the parameters. Bilayer simulations of various lipid headgroups (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol) and tails (saturated, monounsaturated, and polyunsaturated) were performed to validate the model, and significant improvements were seen in bilayer properties, including surface area, membrane thicknesses, NMR deuterium order parameters, and density profiles. C36UAr was also compared to the hydrogen mass repartitioning (HMR) method. The high accuracy and competitive efficiency shown in this study make C36UAr one of the best choices for studies of membrane structure and membrane-associated proteins.

We show that commercially sourced n-channel silicon field-effect transistors (nFETs) operating above their threshold voltage with closed loop feedback to maintain a constant channel current allow a pH readout resolution of (7.2 ± 0.3) × 10 -3 at a bandwidth of 10 Hz, or ≈3-fold better than the open loop operation commonly employed by integrated ion-sensitive field-effect transistors (ISFETs). We leveraged the improved nFET performance to measure the change in solution pH arising from the activity of a pathological form of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, and showed quantitative agreement with previous measurements. The improved pH resolution was realized while the devices were operated in a remote sensing configuration with the pH sensing element off-chip and connected electrically to the FET gate terminal. We compared these results with those measured by using a custom-built dual-gate 2D field-effect transistor (dg2DFET) fabricated with 2D semi-conducting MoS 2 channels and a signal amplification of 8. Under identical solution conditions the nFET performance approached the dg2DFETs pH resolution of (3.9 ± 0.7) × 10 -3 . Finally, using the nFETs, we demonstrated the effectiveness of a custom polypeptide, p5, as a therapeutic agent in restoring the function of Cdk5. We expect that the straight-forward modifications to commercially sourced nFETs demonstrated here will lower the barrier to widespread adoption of these remote-gate devices and enable sensitive bioanalytical measurements for high throughput screening in drug discovery and precision medicine applications.

Ethanolamine plasmalogen (EtnPLA) is a conical-shaped ether lipid and an essential component of neurological membranes. Low stability against oxidation limits its study in experiments. The concentration of EtnPLA in the bilayer varies depending on cell type and disease progression. Here we report on mixed bilayers of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and 1-(1 Z -octadecenyl)-2-oleoyl- sn -glycero-3-phosphoethanolamine (C18(Plasm)-18:1PE, PLAPE), an EtnPLA lipid subtype, at mole ratios of 2:1, 1:1, and 1:2. We present X-ray diffuse scattering (XDS) form factors F ( q z ) from oriented stacks of bilayers, related electron-density profiles, and hydrocarbon chain NMR order parameters. To aid future research on EtnPLA lipids and associated proteins, we have also extended the CHARMM36 all-atom force field to include the PLAPE lipid. The ability of the new force-field parameters to reproduce both X-ray and NMR structural properties of the mixed bilayer is remarkable. Our results indicate a thickening of the bilayer upon incorporation of increasing amounts of PLAPE into mixed bilayers, a reduction of lateral area per molecule, and an increase in lipid tail-ordering. The lateral compressibility modulus ( K A ) calculated from simulations yielded values for PLAPE similar to POPC.

Aided by efforts to improve their speed and efficiency, molecular dynamics (MD) simulations provide an increasingly powerful tool to study the structure-function relationship of pentameric ligand-gated ion channels (pLGICs). However, accurate reporting of the channel state and observation of allosteric regulation by agonist binding with MD remains difficult due to the timescales necessary to equilibrate pLGICs from their artificial and crystalized conformation to a more native, membrane-bound conformation in silico. Here, we perform multiple all-atom MD simulations of the homomeric 5-hydroxytryptamine 3A (5-HT 3A ) serotonin receptor for 15 to 20 μs to demonstrate that such timescales are critical to observe the equilibration of a pLGIC from its crystalized conformation to a membrane-bound conformation. These timescales, which are an order of magnitude longer than any previous simulation of 5-HT 3A , allow us to observe the dynamic binding and unbinding of 5-hydroxytryptamine (5-HT) (i.e., serotonin) to the binding pocket located on the extracellular domain (ECD) and allosteric regulation of the transmembrane domain (TMD) from synergistic 5-HT binding. While these timescales are not long enough to observe complete activation of 5-HT 3A , the allosteric regulation of ion gating elements by 5-HT binding is indicative of a preactive state, which provides insight into molecular mechanisms that regulate channel activation from a resting state. This mechanistic insight, enabled by microsecond-timescale MD simulations, will allow a careful examination of the regulation of pLGICs at a molecular level, expanding our understanding of their function and elucidating key structural motifs that can be targeted for therapeutic regulation., Competing Interests: The authors declare no competing interest.

All-atom models of the long periodicity phase (LPP) in the stratum corneum (SC) are studied using bilayer-slab-bilayer (sandwich) structures and multi-microsecond simulations. Linoleate promotes melting of the interior slab, which contains ceramide (Cer) in the posturing chain conformation, a structurally distinct conformation from full chain extension. The mechanism of Cer transitioning into full extension is characterized by initial anchoring of the head-proximal carbons and occurs over tens of nanoseconds. Free fatty acids translocate through the interior over hundreds of nanoseconds, while Cer and cholesterol take around a microsecond or longer to translocate. Electron density and neutron scattering length density profiles from simulation agree with experiment, and the high disorder of linoleate in CerEOS supports experiments with infrared spectroscopy and nuclear magnetic resonance. Lateral organization demonstrates dependence on lipid composition and bilayer thickness. To further validate the LPP model, umbrella sampling was used to calculate ethanol permeability in comparison with experiment (log( P ) values obtained from modeling the SC's multiple LPP layers are -7.6 and -6.6 cm/s, and that from experiment on cadaver skin is -6.65 cm/s). A "leapfrog" mechanism for ethanol permeation is proposed which is associated with its role as a topical enhancer. These models, the first experimentally verified atomistic sandwich models of the LPP, will aid in the design and optimization of transdermal drug delivery.

We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS 2 , use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10 -6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.

Polysaccharide lyases (PLs) are an important class of proteins that are excreted from bacteria to degrade sugars in the extracellular matrix of the host. The PL from S. maltophilia (Smlt1473) was found to have pH-specific degradation of three varying polysaccharides: alginate, celluronic acid, and hyaluronic acid ( J. Biol. Chem . 2014 , 289 , 18022-18032). In this work, we aim to further understand the effect of pH on sugar binding and cleavage using molecular dynamics (MD) simulations and activity assay experiments. The structure of Smlt1437 was modeled with crystal structure of alginate lyase A1-III (PDB 1QAZ ) from Sphingomonas Sp. Ionizable resides were adjusted based on their predicted p K a 's at pH of 5, 7, and 9 and simulated in solution with predicted docked structures of hyaluronic acid (HA) and poly-β-d-glucuronic acid (poly-GlcUA). These docked structures were simulated for 125 ns using all-atom MD. The simulations with monomers of HA and poly-GlcUA revealed the importance of loop1 of the protein to cover the binding pocket. Glu212 was found to be important with cross-domain interactions of loop1 to stabilize a closed binding pocket state. Mutations of this residue (E212G) resulted in significant reduction of enzyme activity compared to the wildtype, which our simulations predict prevents the formation of a stable salt bridge with Arg215. Asp48 on loop1 appears to limit the activity of this enzyme as the D48G mutant shows an increased activity, which based on our MD simulations suggest that Asp48 interactions can block sugar binding with Arg218. MD simulations of the HA 4mer resulted in consistent protein interactions that stabilize sugar binding to Smlt1473 and provide insight into the importance of Trp171 to binding. Overall, this joint experimental/computational study has probed allosteric effects of sugar binding to loop1 stability that influences enzyme activity.

Metal-mediated exfoliation has been demonstrated as a promising approach for obtaining large-area flakes of two-dimensional (2D) materials to fabricate prototypical nanoelectronics. However, several processing challenges related to organic contamination at the interface of a 2D material and gate oxide must be overcome to realize robust devices with high yields. Here, we demonstrate an optimized process to realize high-performance field-effect transistor (FET) arrays from large-area (∼5000 μm 2 ), monolayer MoS 2 with a yield of 85%. A central element of this process is an exposed material forming gas anneal (EM-FGA) that results in uniform FET performance metrics (i.e., field-effect mobilities, threshold voltages, and contact performance). Complementary analytical measurements show that the EM-FGA process reduces deleterious channel doping effects by decreasing organic contamination while also reducing the prevalence of insulating molybdenum oxide, effectively improving the MoS 2 -gate oxide interface. The uniform FET performance metrics and high device yield achieved by applying the EM-FGA technique on large-area 2D material flakes will help advance the fabrication of complex 2D nanoelectronic devices and demonstrate the need for improved engineering of the 2D material-gate oxide interface.

The amphipathic nature of the lipid molecule (hydrophilic head and hydrophobic tails) enables it to act as a barrier between fluids with various properties and to sustain an environment where the processes critical to life may proceed. While computer simulations of biomolecules primarily investigate protein conformation and binding to drug-like molecules, these interactions often occur in the context of a lipid membrane. Chemical specificity of lipid models is essential to accurately represent the complex environment of the lipid membrane. This review discusses the development and performance of currently used chemically specific lipid force fields (FF) such as the CHARMM, AMBER, GROMOS, OPLS, and MARTINI families. Considerations in lipid FF development including lipid diversity, temperature dependence, phase behavior, and effects of atomic polarizability are considered, as well as methods and goals of parametrization. Applications of these FFs to complex and diverse models for cellular membranes are summarized. Lastly, areas for future development, such as efficient inclusion of long-range Lennard-Jones interactions (significant in transitions from polar to apolar media), accurate transmembrane dipole potential, and diffusion under periodic boundary conditions are considered.

The stratum corneum (SC), which functions as a barrier to many exogenous molecules, contains ceramides (Cer) in both bilayer and multilayer structures. Simple models of the SC using pure Cer lamellae are simulated and studied using CHARMM36 force field. For single bilayers, the chain length is noted to have a significant effect on the thickness and interdigitation, whereas hydroxylation induces more prominent effects in hydrogen bonding. CerEOS, due to its long fatty acid tail (48 carbons), is simulated as a double layer that possesses the potential to transition into an interior-disordered state, which is reminiscent of the sandwich model of the long periodicity phase of the SC. The surface area per lipid of CerEOS is significantly higher than in other Cer bilayers, which affects lateral properties such as clustering. Compared to Cer bilayers, CerEOS chains are similarly ordered except in the disordered linoleate, and interdigitation decreases in disordered CerEOS. Umbrella sampling simulations are performed on bilayers of sphingosine Cer to calculate the potential of mean force (PMF) for ethanol penetration as well as the permeability. The chain length is noted to both cause an outward shift of the PMF as well as an order-of-magnitude decrease in permeability, although the bilayer center is still disordered and favorable for permeation. The structures probed in this work will guide and serve as control comparisons to more complex simulations of accurate SC mixtures.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Nanodiscs are discoidal protein-lipid complexes that have wide applications in membrane protein studies. Modeling and simulation of nanodiscs are challenging due to the absence of structures of many membrane scaffold proteins (MSPs) that wrap around the membrane bilayer. We have developed CHARMM-GUI Nanodisc Builder (http://www.charmm-gui.org/input/nanodisc) to facilitate the setup of nanodisc simulation systems by modeling the MSPs with defined size and known structural features. A total of 11 different nanodiscs with a diameter from 80 to 180 Å are made available in both the all-atom CHARMM and two coarse-grained (PACE and Martini) force fields. The usage of the Nanodisc Builder is demonstrated with various simulation systems. The structures and dynamics of proteins and lipids in these systems were analyzed, showing similar behaviors to those from previous all-atom and coarse-grained nanodisc simulations. We expect the Nanodisc Builder to be a convenient and reliable tool for modeling and simulation of nanodisc systems. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)