壳聚糖与细菌细胞膜相互作用的分子动力学研究

目的 壳聚糖（chitosan,CS）是一种天然的广谱抗菌活性物质。现有研究表明,壳聚糖与细菌细胞膜的相互作用是其发挥抗菌功能的关键。受限于传统实验技术的表征能力,壳聚糖与细菌细胞膜相互作用的具体机制仍有待研究。本文旨在研究壳聚糖与细菌细胞膜相互作用的分子机制。方法 本研究利用全原子分子动力学模拟技术主要探究了完全脱乙酰化的不同聚合度壳聚糖（八聚糖、十二聚糖和十六聚糖）与革兰氏阴性菌外膜（outer membrane,OM）和革兰氏阳性菌质膜（cytoplasmic membrane,CM）相互作用的动态过程。结果 壳聚糖主要依靠其氨基、碳6位羟基和碳3位羟基与OM和CM的头部极性区发生快速结合。随后壳聚糖末端糖基单元倾向于插入OM内部,深度约1 nm,并与脂质分子脂肪酸链上的羰基形成稳定的氢键相互作用。与之相比,壳聚糖分子难以稳定地插入CM内部。壳聚糖结合对膜结构性质产生影响,主要表现在降低OM和CM的单分子脂质面积,显著减少OM和CM极性区的Ca2+和Na+数目,破坏阳离子介导的脂质间相互作用。结论 本研究证明,壳聚糖带正电的氨基基团是介导其与膜相互作用的关键,并破环脂质间的相互作...     

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.     

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.     

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.     

Characterizing Membrane Association and Periplasmic Transfer of Bacterial Lipoproteins through Molecular Dynamics Simulations

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Subcellular Localization of Gram-Negative Bacterial Proteins Using Sparse Learning

One of the main challenges faced by biological applications is to predict protein subcellular localization in an automatic fashion accurately. To achieve this in these applications, a wide variety of machine learning methods have been proposed in recent years. Most of them focus on finding the optimal classification scheme and less of them take the simplifying the complexity of biological system into account. Traditionally such bio-data are analyzed by first performing a feature selection before classification. Motivated by CS (Compressive Sensing), we propose a method which performs locality preserving projection with a sparseness criterion such that the feature selection and dimension reduction are merged into one analysis. The proposed sparse method decreases the complexity of biological system, while increases protein subcellular localization accuracy. Experimental results are quite encouraging, indicating that the aforementioned sparse method is quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gram-negative bacterial proteins.     

细菌鞭毛马达——一种卓越的分子机器

鞭毛马达(flagellar motor)是一种分子旋转马达,它在细菌鞭毛的结构与功能中起着中心作用.鞭毛马达的结构已基本清楚,主要由Mot A、Mot B、Fli G、Fli M和Fli N 5种蛋白组成定子(stator)和转子(rotor),其驱动力来自于跨膜的H⁺或Na⁺流.目前对鞭毛马达的旋转动力学及旋转力矩产生机制已有初步的了解.鞭毛马达可作为研究分子旋转马达的理想模型,对其深入研究将有助于认识生物能量转化利用及细胞运动的机制并具有广泛的生物学意义.     

ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition

The rotational surveillance and energy transfer (ROSET) model of TonB action suggests a mechanism by which the electrochemical proton gradient across the Gram-negative bacterial inner membrane (IM) promotes the transport of iron through ligand-gated porins (LGP) in the outer membrane (OM). TonB associates with the IM by an N-terminal hydrophobic helix that forms a complex with ExbBD. It also contains a central extended length of rigid polypeptide that spans the periplasm and a dimeric C-terminal-ββαβ-domain (CTD) with LysM motifs that binds the peptidoglycan (PG) layer beneath the OM bilayer. The TonB CTD forms a dimer with affinity for both PG- and TonB-independent OM proteins (e.g., OmpA), localizing it near the periplasmic interface of the OM bilayer. Porins and other OM proteins associate with PG, and this general affinity allows the TonB CTD dimer to survey the periplasmic surface of the OM bilayer. Energized rotational motion of the TonB N terminus in the fluid IM bilayer promotes the lateral movement of the TonB-ExbBD complex in the IM and of the TonB CTD dimer across the inner surface of the OM. When it encounters an accessible TonB box of a (ligand-bound) LGP, the monomeric form of the CTD binds and recruits it into a 4-stranded β-sheet. Because the CTD is rotating, this binding reaction transfers kinetic energy, created by the electrochemical proton gradient across the IM, through the periplasm to the OM protein. The equilibration of the TonB C terminus between the dimeric and monomeric forms that engage in different binding reactions allows the identification of iron-loaded LGP and then the internalization of iron through their trans-outer membrane β-barrels. Hence, the ROSET model postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake.     

Complement Activation by Bacterial Surface Glycolipids: A Study with Planar Bilayer Membranes

Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd₂ LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent on the presence of C9, indicating that the observed current changes were due to C activation. We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd₂ LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1 and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway. Received: 16 July 1998/Revised: 28 October 1998     

MD Simulations of a 5-HT2A Receptor Model in DOPC Membranes

Abstract

This work presents MD calculations on a model 5-HT_2A G protein-coupled receptor embedded in DOPC membrane bilayers at different lipid:protein ratios. The primary purpose is to evaluate physical properties of the system to probe membrane dynamics and the solvation interactions. This showed several kinds of apparent cooperativity phenomena in distributions, lipid dynamics, and hydrogen bond interactions. The integral protein appears to cause a disordered condensation of the local lipid structure which does not extend far beyond the surface layer of lipids. This, and changes in the lipid-protein interaction profiles give a start toward understanding membrane-protein selectivity. The presence of a partially immobilized surface layer of lipids may generally slow down kinetics of solute-solute interactions in similar liquid-crystal membranes. A hydration algorithm was also applied to the GPCR model producing a detailed water structure in some of the internal cavities, including in the areas proposed for ligand binding. Other properties examined included the distributions of lipid groups, the membrane electrostatic potential, and some of the short-time protein dynamics.     

Plasposons: Modular Self-Cloning Minitransposon Derivatives for Rapid Genetic Analysis of Gram-Negative Bacterial Genomes

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon’s site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.     

Computer Simulations of Protein Diffusion in Compartmentalized Cell Membranes

The diffusion of proteins in the cell membrane is investigated using computer simulations of a two-dimensional model. The membrane is assumed to be divided into compartments, with adjacent compartments separated by a barrier of stationary obstacles. Each compartment contains traps represented by stationary attractive disks. Depending on their size, these traps are intended to model either smaller compartments or binding sites. The simulations are intended to model the double-compartment model, which has been used to interpret single molecule experiments in normal rat kidney cells, where five regimes of transport are observed. The simulations show, however, that five regimes are observed only when there is a large separation between the sizes of the traps and large compartments, casting doubt on the double compartment model for the membrane. The diffusive behavior is sensitive to the concentration and size of traps and the strength of the barrier between compartments suggesting that the diffusion of proteins can be effectively used to characterize the structure of the membrane.     

Monte Carlo Simulations of Vesicles and Fluid Membranes Transformations

The appearance of compartmentalization is recognized as a key step in biogenesis. The study of the dynamical behaviour of amphiphilic close membranes at equilibrium or under some external stress (osmotic pressure or dehydration process) can be useful in order to better elucidate the role of vesicles in the origin of life and to get insight into the molecular and membrane properties that bring to a spontaneous vesicle division. A Monte Carlo approach to simulate the evolution of close membranes under an external stress will be presented. This approach is mainly based on the accepted surface energy model introduced by Helfrich (1973) and Seifert (1997a). Some preliminary results will be also illustrated and possible developments and limits of this method discussed.     

MDN: A Web Portal for Network Analysis of Molecular Dynamics Simulations

We introduce a web portal that employs network theory for the analysis of trajectories from molecular dynamics simulations. Users can create protein energy networks following methodology previously introduced by our group, and can identify residues that are important for signal propagation, as well as measure the efficiency of signal propagation by calculating the network coupling. This tool, called MDN, was used to characterize signal propagation in Escherichia coli heat-shock protein 70-kDa. Two variants of this protein experimentally shown to be allosterically active exhibit higher network coupling relative to that of two inactive variants. In addition, calculations of partial coupling suggest that this quantity could be used as part of the criteria to determine pocket druggability in drug discovery studies.     

Gibbs-Ensemble Molecular Dynamics: A New Method for Simulations Involving Particle Exchange

We discuss a novel simulation method suitable for simulating phenomena involving particle exchange. The method is a molecular dynamics version of the Gibbs-Ensemble Monte Carlo technique, which has been developed some years ago for the direct simulation of phase equilibria in fluid systems. The idea is to have two separate simulation boxes, which can exchange particles or molecules in a thermodynamically consistent fashion. We discuss the general idea of the Gibbs-Ensemble Molecular Dynamics technique and present examples for different simple atomic and molecular fluids. Specifically we will discuss Gibbs-Ensemble Molecular Dynamics simulations of gas-liquid and liquid-solid equilibria in Lennard-Jones systems and in hexane as well as an application of the method to adsorption.     

Short-Term Starvation of Immune Deficient Drosophila Improves Survival to Gram-Negative Bacterial Infections

Background

Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-κB) pathway. Since the TIR-NF-κB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions.

Methodology/Principal Findings

We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-κB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-κB protein Dif, usually needed for responses against Gram-positive bacteria.

Conclusions/Significance

Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.     

Nanostructured Biosensors Based on Components of Bacterial Membranes

Biophysics - Protein molecules have a unique property of transferring material or information across the impenetrable barrier of a lipid bilayer. This peculiar role of cell...