The effects of the lipid type on the spatial arrangement and dynamics of cholesterol in binary component lipid membranes.

Intermolecular interactions between cholesterol and lipids in cell membranes, which play critical roles in cellular processes such as the formation of nano-domains, depend on the molecular structure of the lipids. The diffusion and the spatial arrangement of cholesterol within the lipid membranes also change with the type of lipids. For example, the flip-flop, an important transport mechanism for cholesterol in the membranes, can be facilitated significantly by the presence of unsaturated lipids. However, how the structure of lipids affects the spatial arrangement and the dynamics of cholesterol remains elusive at a molecular level. In this study, we investigate the effects of lipid-cholesterol interactions on the spatial arrangement and the dynamics of cholesterol. We perform molecular dynamics simulations for the binary component membranes of lipids and cholesterol. We employ seven different kinds of lipids by changing either the degree of a saturation level or the length of lipid tails. We find from our simulations that the rate of cholesterol flip-flop is enhanced as the lipids are either less saturated or shorter, which is consistent with previous studies. Interestingly, when the lipid tails are fully saturated and sufficiently long, the center in between two leaflets becomes metastable for cholesterol to stay at. Because the cholesterol at the membrane center diffuses faster than that within leaflets, regardless of the lipid type, such an emergence of the metastable state (in terms of the cholesterol position) complicates the cholesterol diffusion significantly.

The spatial arrangement of a single nanoparticle in a thin polymer film and its effect on the nanoparticle diffusion.

The spatial arrangement of nanoparticles (NPs) within thin polymer films may influence their properties such as the glass transition temperature. Questions regarding what may affect the spatial arrangement of NPs, however, still remain unanswered at a molecular level. In this work, we perform molecular dynamics simulations for a free-standing thin polymer film with a single NP. We find from simulations that depending on the NP size and the inter-particle interaction between the NP and polymers, one may control the spatial arrangement of the NP. When the interaction between the NP and polymers is sufficiently attractive (repulsive), the NP is likely to be placed at the center (at the surface) of the thin film in equilibrium. Interestingly, for a moderate interaction between the NP and polymers, the first-order transition occurs in the spatial arrangement of the NP as one increases the NP size: a small NP prefers the surface of the polymer film whereas a large NP prefers the center. Such a first-order transition is corroborated by calculating the free energy of the NP as a function of the position and can be understood in terms of a sixth-order Landau free energy. More interestingly, the diffusion of the NP also changes drastically due to the first-order transition in the spatial arrangement. The NP diffusion is enhanced drastically (more than expected in bulk polymer melts) as the NP is shifted to the polymer film surface.

2-Acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2.

Chitin, a homopolymer of ß1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N'-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.

Non-equilibrium transport of nanoparticles across the lipid membrane.

Development of effective strategies for the internalization of nanoparticles is essential in many applications, such as drug delivery. Most, if not all, previous studies are based on equilibrium considerations. In this work, inspired by the recent development of a pro-drug delivery strategy based on reversible esterification, we consider a non-equilibrium transport mechanism for nanoparticles of a 6 nm diameter across the lipid membrane. We divide the transport process into insertion and ejection steps, which are studied with coarse-grained models using free energy and reactive Monte Carlo simulations, respectively. The simulations show that the non-equilibrium transport efficiency is relatively insensitive to the fraction of reactive surface ligands once a modest threshold is surpassed, while the distribution pattern of different (hydrophilic, reactive and permanent hydrophobic) ligands on the nanoparticle surface has a notable impact on both the insertion and ejection steps. Our study thus supports a novel avenue for designing nanoparticles that are able to be efficiently internalized and provides a set of relevant guidelines for surface functionalization.

Cholesterol-Mediated Clustering of the HIV Fusion Protein gp41 in Lipid Bilayers.

The envelope glycoprotein (Env) of the human immunodeficient virus (HIV-1) is known to cluster on the viral membrane surface to attach to target cells and cause membrane fusion for HIV-1 infection. However, the molecular structural mechanisms that drive Env clustering remain opaque. Here, we use solid-state NMR spectroscopy and molecular dynamics (MD) simulations to investigate nanometer-scale clustering of the membrane-proximal external region (MPER) and transmembrane domain (TMD) of gp41, the fusion protein component of Env. Using 19F solid-state NMR experiments of mixed fluorinated peptides, we show that MPER-TMD trimers form clusters with interdigitated MPER helices in cholesterol-containing membranes. Inter-trimer 19F-19F cross peaks, which are indicative of spatial contacts within ∼2 nm, are observed in cholesterol-rich virus-mimetic membranes but are suppressed in cholesterol-free model membranes. Water-peptide and lipid-peptide cross peaks in 2D 1H-19F correlation spectra indicate that the MPER is well embedded in model phosphocholine membranes but is more exposed to the surface of the virus-mimetic membrane. These experimental results are reproduced in coarse-grained and atomistic molecular dynamics simulations, which suggest that the effects of cholesterol on gp41 clustering is likely via indirect modulation of the MPER orientation. Cholesterol binding to the helix-turn-helix region of the MPER-TMD causes a parallel orientation of the MPER with the membrane surface, thus allowing MPERs of neighboring trimers to interact with each other to cause clustering. These solid-state NMR data and molecular dynamics simulations suggest that MPER and cholesterol cooperatively govern the clustering of gp41 trimers during virus-cell membrane fusion.

Electronic Structure of de Novo Peptide ACC-Hex from First Principles.

Proteins are promising components for bioelectronic devices due in part to their biocompatibility, flexibility, and chemical diversity, which enable tuning of material properties. Indeed, an increasingly broad range of conductive protein supramolecular materials have been reported. However, due to their structural and environmental complexity, the electronic structure, and hence conductivity, of protein assemblies is not well-understood. Here we perform an all-atom simulation of the physical and electronic structure of a recently synthesized self-assembled peptide antiparallel coiled-coil hexamer, ACC-Hex. Using classical molecular dynamics and first-principles density functional theory, we examine the interactions of each peptide, containing phenylalanine residues along a hydrophobic core, to form a hexamer structure. We find that while frontier electronic orbitals are composed of phenylalanine, the peptide backbone and remaining residues, including those influenced by solvent, also contribute to the electronic density. Additionally, by studying dimers extracted from the hexamer, we show that structural distortions due to atomic fluctuations significantly impact the electronic structure of the peptide bundle. These results indicate that it is necessary to consider the full atomistic picture when using the electronic structure of supramolecular protein complexes to predict electronic properties.

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast.

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.

Interdomain exchange and the flip-flop of cholesterol in ternary component lipid membranes and their effects on heterogeneous cholesterol diffusion.

Cell membranes are heterogeneous with a variety of lipids, cholesterol, and proteins and are composed of domains of different compositions. Such heterogeneous environments make the transport of cholesterol complicated: cholesterol not only diffuses within a particular domain but also travels between domains. Cholesterol also flip-flops between upper and lower leaflets such that cholesterol may reside both within leaflets and in the central region between two leaflets. How the presence of multiple domains and the interdomain exchange of cholesterol would affect the cholesterol transport, however, remains elusive. In this study, therefore, we perform molecular dynamics simulations up to 100µs for ternary component lipid membranes, which consist of saturated lipids (dipalmitoylphosphatidylcholine, DPPC), unsaturated lipids (dilinoleylphosphatidylcholine, DIPC), and cholesterol. The ternary component membranes in our simulations form two domains readily: DPPC and DIPC domains. We find that the diffusion of cholesterol molecules is much more heterogeneous and non-Gaussian than expected for binary component lipid membranes of lipids and cholesterol. The non-Gaussian parameter of the cholesterol molecules is about four times larger in the ternary component lipid membranes than in the binary component lipid membranes. Such non-Gaussian and heterogeneous transport of cholesterol arises from the interplay among the interdomain kinetics, the different diffusivity of cholesterol in different domains, and the flip-flop of cholesterol. This suggests that in cell membranes that consist of various domains and proteins, the cholesterol transport can be very heterogeneous. We also find that the mechanism of the interdomain exchange differs for different domains: cholesterol tends to exit the DIPC domain along the central region of the membrane for the DIPC-to-DPPC transition, while the cholesterol is likely to exit the DPPC domain within the membrane leaflet for the DPPC-to-DIPC transition. Also, the interdomain exchange kinetics of cholesterol for the DPPC-to-DIPC transition is up to 7.9 times slower than the DIPC-to-DPPC transition.

Translation-rotation decoupling of tracers reflects medium-range crystalline order in two-dimensional colloid glasses.

The dynamic heterogeneity and the translation-rotation decoupling are the dynamic signatures of glasses and supercooled liquids. Whether and how the dynamic heterogeneity would relate to the local structure of glasses has been a puzzle for decades. In this work we perform molecular dynamics simulations for tracers in both two-dimensional polydisperse colloids (2DPC) and two-dimensional binary colloids (2DBC). In 2DPC glasses, hexatic local structures develop at low enough temperatures and grow quickly along with the dynamic correlation length of the 2DPC, which is well known as the medium-range crystalline order (MRCO). In 2DBC glasses, on the other hand, any explicit local structure has not been reported to grow significantly with the dynamic correlation length at low temperatures. We introduce two different types of tracers into colloidal systems: A diamond tracer that resembles the MRCO of 2DPC glasses and a square tracer that is dissimilar to any local structure of glasses. The translation-rotation decoupling of the diamond tracer in 2DPC glasses is much more significant than that of the square tracer in the same 2DPC glasses. On the other hand, such a tracer shape-dependence of the decoupling is not observed in 2DBC glasses where the local hexatic structure does not develop significantly. We introduce a shape-dependency parameter of the decoupling and find that the shape-dependency parameter grows along with the dynamic correlation length in 2DPC glasses but not in 2DBC glasses. This illustrates that the dynamic heterogeneity and the translation-rotation decoupling of tracers could reveal the local structure that develops in glasses.

Cholesterol Interaction with the Trimeric HIV Fusion Protein gp41 in Lipid Bilayers Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

HIV-1 entry into cells is mediated by the fusion protein gp41. Cholesterol plays an important role in this virus-cell fusion, but molecular structural information about cholesterol-gp41 interaction is so far absent. Here, we present experimental and computational data about cholesterol complexation with gp41 in lipid bilayers. We focus on the C-terminal region of the protein, which comprises a membrane-proximal external region (MPER) and the transmembrane domain (TMD). We measured peptide-cholesterol contacts in virus-mimetic lipid bilayers using solid-state NMR spectroscopy, and augmented these experimental data with all-atom molecular dynamics simulations. 2D 19F NMR spectra show correlation peaks between MPER residues and the cholesterol isooctyl tail, indicating that cholesterol is in molecular contact with the MPER-TMD trimer. 19F-13C distance measurements between the peptide and 13C-labeled cholesterol show that C17 on the D ring and C9 at the intersection of B and C rings are ~7.0 Å from the F673 side-chain 4-19F. At high peptide concentrations in the membrane, the 19F-13C distance data indicate three cholesterol molecules bound near F673 in each trimer. Mutation of a cholesterol recognition amino acid consensus motif did not change these distances, indicating that cholesterol binding does not require this sequence motif. Molecular dynamics simulations further identify two hotspots for cholesterol interactions. Taken together, these experimental data and simulations indicate that the helix-turn-helix conformation of the MPER-TMD is responsible for sequestering cholesterol. We propose that this gp41-cholesterol interaction mediates virus-cell fusion by recruiting gp41 to the boundary of the liquid-disordered and liquid-ordered phases to incur membrane curvature.

Artificial Intracellular Filaments.

Intracellular protein filaments are ubiquitous for cellular functions, but forming bona fide biomimetic intracellular filaments of small molecules in living cells remains elusive. Here, we report the in situ formation of self-limiting intracellular filaments of a small peptide via enzymatic morphological transition of a phosphorylated and trimethylated heterochiral tetrapeptide. Enzymatic dephosphorylation reduces repulsive intermolecular electrostatic interactions and converts the peptidic nanoparticles into filaments, which exhibit distinct types of cross-ß structures with either C7 or C2 symmetries, with the hydrophilic C-terminal residues at the periphery of the helix. Macromolecular crowding promotes the peptide filaments to form bundles, which extend from the plasma membrane to nuclear membrane and hardly interact with endogenous components, including cytoskeletons. Stereochemistry and post-translational modification (PTM) of peptides are critical for generating the intracellular bundles. This work may offer a way to gain lost functions or to provide molecular insights for understanding normal and aberrant intracellular filaments.

Position-Dependent Diffusion Dynamics of Entangled Polymer Melts Nanoconfined by Parallel Immiscible Polymer Films.

The morphological structure and dynamics of confined polymers adjacent to the polymer-polymer interface have a profound effect on determining the overall physical properties of polymer blends. We measured the diffusion dynamics of poly(methyl methacrylate) (PMMA) melts confined between polystyrene (PS) layers using neutron reflectivity. Combinations of various thicknesses of PMMA and deuterated PMMA (dPMMA) allowed us to experimentally reveal the nonmonotonic behavior of polymer mobility near the PS-PMMA interface. From the neutron reflectivity results, we found that the polymers adjacent to the immiscible polymer-polymer interface showed enhanced diffusion dynamics because of the repulsive interaction between PS and PMMA, whereas the polymer at local regions farther from the interface exhibited reduced dynamics. This is probably due to the nonspherical conformation of PMMA and spatial confinement near the PS-PMMA interface.

Enhanced Dynamics of Confined Polymers near the Immiscible Polymer-Polymer Interface: Neutron Reflectivity Studies.

For polymer-blend films, local dynamics in confined polymer domains tend to differ from the bulk because of significant contributions from the polymer-polymer interface. Herein, we investigated the diffusion dynamics of entangled polymer thin films confined between different polymers in a direction perpendicular to the surface using neutron reflectivity. We found that a bilayer of poly(methyl methacrylate) (PMMA) and deuterated PMMA (dPMMA) sandwiched between polystyrene (PS) layers exhibited significant increase in mobility near the polymer-polymer interface with decreasing PMMA thickness. This indicates that the contribution of repulsive interactions at the immiscible polymer-polymer interface becomes more significant as the film thickness decreases. We also found that the interfacial roughness between PMMA and PS (28 Å at equilibrium) and soft confinement of PS layers did not significantly affect the change in the diffusion dynamics of the adjacent PMMA. This was evidenced by comparison with the diffusion results of multilayers with a flat interface (8 Å at equilibrium) between PMMA and hard PS by UV cross-linking.

Spatial Dependence of Non-Gaussian Diffusion of Nanoparticles in Free-Standing Thin Polymer Films.

The addition of nanoparticles (NPs) to a free-standing polymer film affects the properties of the film such as viscosity and glass transition temperature. Recent experiments, for example, showed that the glass transition temperature of thin polymer films was dependent on how NPs were distributed within the polymer films. However, the spatial arrangement of NPs in free-standing polymer films and its effect on the diffusion of NPs and polymers remain elusive at a molecular level. In this study, we employ generic coarse-grained models for polymers and NPs and perform extensive molecular dynamics simulations to investigate the diffusion of polymers and NPs in free-standing thin polymer films. We find that small NPs are likely to stay at the interfacial region of the polymer film, while large NPs tend to stay at the center of the film. On the other hand, as the interaction between a NP and a monomer becomes more attractive, the NP is more likely to be placed at the film center. The diffusion of monomers slows down slightly as more NPs are added to the film. Interestingly, the NP diffusion is dependent strongly on the spatial arrangement of the NPs: NPs at the interfacial region diffuse faster and undergo more non-Gaussian diffusion than NPs at the film center, which implies that the interfacial region would be more mobile and dynamically heterogeneous than the film center. We also find that the mechanism for non-Gaussian diffusion of NPs at the film center differs from that at the interfacial region and that the NP diffusion would reflect the local viscosity of the polymer films.

Facilitated and Non-Gaussian Diffusion of Cholesterol in Liquid Ordered Phase Bilayers Depends on the Flip-Flop and Spatial Arrangement of Cholesterol.

The diffusion of cholesterol in biological membranes is critical to cellular processes such as the formation of cholesterol-enriched domains. The cholesterol diffusion may be complicated especially when cholesterol flip-flops and/or stays at the membrane center. Understanding the diffusion mechanism of cholesterol at a molecular level should be, therefore, a topic of interest. We perform molecular dynamics simulations up to 100 µs for lipid bilayers with various concentrations of cholesterol. We find that cholesterol diffusion in the liquid ordered phase depends on whether it is within leaflets or at the bilayer center, is non-Gaussian for several microseconds, and is enhanced significantly compared to that of lipids. Cholesterol at the bilayer center diffuses fast, while cholesterol in the hydrocarbon region with upright orientation diffuses relatively slowly. Such position-dependent dynamics of cholesterol leads to facilitated and non-Gaussian diffusion.

Hierarchically self-assembled hexagonal honeycomb and kagome superlattices of binary 1D colloids.

Synthesis of binary nanoparticle superlattices has attracted attention for a broad spectrum of potential applications. However, this has remained challenging for one-dimensional nanoparticle systems. In this study, we investigate the packing behavior of one-dimensional nanoparticles of different diameters into a hexagonally packed cylindrical micellar system and demonstrate that binary one-dimensional nanoparticle superlattices of two different symmetries can be obtained by tuning particle diameter and mixing ratios. The hexagonal arrays of one-dimensional nanoparticles are embedded in the honeycomb lattices (for AB2 type) or kagome lattices (for AB3 type) of micellar cylinders. The maximization of free volume entropy is considered as the main driving force for the formation of superlattices, which is well supported by our theoretical free energy calculations. Our approach provides a route for fabricating binary one-dimensional nanoparticle superlattices and may be applicable for inorganic one-dimensional nanoparticle systems.Binary mixtures of 1D particles are rarely observed to cooperatively self-assemble into binary superlattices, as the particle types separate into phases. Here, the authors design a system that avoids phase separation, obtaining binary superlattices with different symmetries by simply tuning the particle diameter and mixture composition.

Hof1 and Chs4 Interact via F-BAR Domain and Sel1-like Repeats to Control Extracellular Matrix Deposition during Cytokinesis.

Localized extracellular matrix (ECM) remodeling is thought to stabilize the cleavage furrow and maintain cell shape during cytokinesis [1-14]. This remodeling is spatiotemporally coordinated with a cytoskeletal structure pertaining to a kingdom of life, for example the FtsZ ring in bacteria [15], the phragmoplast in plants [16], and the actomyosin ring in fungi and animals [17, 18]. Although the cytoskeletal structures have been analyzed extensively, the mechanisms of ECM remodeling remain poorly understood. In the budding yeast Saccharomyces cerevisiae, ECM remodeling refers to sequential formations of the primary and secondary septa that are catalyzed by chitin synthase-II (Chs2) and chitin synthase-III (the catalytic subunit Chs3 and its activator Chs4), respectively [18, 19]. Surprisingly, both Chs2 and Chs3 are delivered to the division site at the onset of cytokinesis [6, 20]. What keeps Chs3 inactive until secondary septum formation remains unknown. Here, we show that Hof1 binds to the Sel1-like repeats (SLRs) of Chs4 via its F-BAR domain and inhibits Chs3-mediated chitin synthesis during cytokinesis. In addition, Hof1 is required for rapid accumulation as well as efficient removal of Chs4 at the division site. This study uncovers a mechanism by which Hof1 controls timely activation of Chs3 during cytokinesis and defines a novel interaction and function for the conserved F-BAR domain and SLR that are otherwise known for their abilities to bind membrane lipids [21, 22] and scaffold protein complex formation [23].

Swing motion as a diffusion mechanism of lipid bilayers in a gel phase.

Lipid bilayers are a model system for studying the properties of cell membranes. For lipid bilayers of a single lipid component, there is a phase transition from a fluid phase to a gel phase as the temperature is decreased. The dynamic behavior of lipids in the gel phase is interesting: some models show dynamic heterogeneity with a large disparity in timescales between fast and slow molecules, and a spatial segregation of the slow molecules. In this paper we study the dynamics of coarse-grained models of lipid bilayers using the dry Martini, Lennard-Jones Martini, polarizable Martini, and BMW models. All four models show similar dynamical behaviors in the gel phase although the transition temperature is model-dependent. We find that the primary mode of transport in the gel phase is a hopping of the lipid molecules. Hopping is seen in both the translational and rotational dynamics, which are correlated, i.e., the lipid molecules display a swing-like motion in the gel phase.

Fermentative l-lactic acid production from pretreated whole slurry of oil palm trunk treated by hydrothermolysis and subsequent enzymatic hydrolysis.

A simple and cost-effective biochemical conversion process consisting of hydrothermal treatment, enzymatic hydrolysis and fermentation of pretreated whole slurry (PWS) was developed for producing l-lactic acid (L-LA) from oil palm trunk (OPT). When OPT was hydrothermally treated at optimal condition capable of achieving maximum yield of hemicellulosic sugars after enzymatic hydrolysis, the enzymatic digestibility of the PWS afforded a yield of 81.4% of the theoretical glucose yield (TGY). However, glucose yield from washed pretreated solid (WPS) was only 43.5% of TGY. The use of two hydrolysates from PWS and WPS for fermentation by Lactobacillus paracasei engineered to selectively produce L-LA afforded yields of 89.5% and 45.8% of the theoretical LA yield (TLY), respectively. This study confirmed the inevitable extensive sugar loss during washing of pretreated slurry due to loss of soluble starch. Alternatively, the proposed design process is considered suitable for converting OPT to L-LA without such starch loss.

Fabrication of an integrated optical filter using a large-core multimode waveguide vertically coupled to a single-mode waveguide.

We demonstrate the feasibility of the process for fabricating a single-mode waveguide and a large-core multimode waveguide aligned vertically on the same substrate. Using this process, we propose and demonstrate a filter that drops optical signal propagating in a single-mode waveguide to a multimode waveguide in the specific wavelength interval by a long-period grating. We use perfluorocyclobutane and benzocyclobutane for the cladding and core of the single-mode waveguide, respectively. The large core of the multimode waveguide is made of Norland Optical Adhesive 61. For the grating period of 315.9 um, the fabricated filter has the center wavelength of 1537.7 nm, at which the maximum attenuation is 17.8 dB.