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...     

Dynamics of Bcl-xL in Water and Membrane: Molecular Simulations

The Bcl2 family of proteins is capable of switching the apoptotic machinery by directly controlling the release of apoptotic factors from the mitochondrial outer membrane. They have ‘pro’ and ‘anti’-apoptotic subgroups of proteins which antagonize each other’s function; however a detailed atomistic understanding of their mechanisms based on the dynamical events, particularly in the membrane, is lacking. Using molecular dynamics simulations totaling 1.6µs we outline the major differences between the conformational dynamics in water and in membrane. Using implicit models of solvent and membrane, the simulated results reveal a picture that is in agreement with the ‘hit-and run’ concept which states that BH3-only peptides displace the tail (which acts as a pseudo substrate of the protein itself) from its binding pocket; this helps the membrane association of the protein after which the BH3 peptide becomes free. From simulations, Bcl-xL appears to be auto-inhibited by its C-terminal tail that embeds into and covers the hydrophobic binding pocket. However the tail is unable to energetically compete with BH3-peptides in water. In contrast, in the membrane, neither the tail nor the BH3-peptides are stable in the binding pocket and appear to be easily dissociated off as the pocket expands in response to the hydrophobic environment. This renders the binding pocket large and open, thus receptive to interactions with other protein partners. Principal components of the motions are dramatically different in the aqueous and in the membrane environments and provide clues regarding the conformational transitions that Bcl-xL undergoes in the membrane, in agreement with the biochemical data.     

Energetic Changes Caused by Antigenic Module Insertion in a Virus-Like Particle Revealed by Experiment and Molecular Dynamics Simulations

The success of recombinant virus-like particles (VLPs) for human papillomavirus and hepatitis B demonstrates the potential of VLPs as safe and efficacious vaccines. With new modular designs emerging, the effects of antigen module insertion on the self-assembly and structural integrity of VLPs should be clarified so as to better enabling improved design. Previous work has revealed insights into the molecular energetics of a VLP subunit, capsomere, comparing energetics within various solution conditions known to drive or inhibit self-assembly. In the present study, molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were performed to examine the molecular interactions and energetics in a modular capsomere of a murine polyomavirus (MPV) VLP designed to protect against influenza. Insertion of an influenza antigenic module is found to lower the binding energy within the capsomere, and a more active state is observed in Assembly Buffer as compared with that in Stabilization Buffer, which has been experimentally validated through measurements using differential scanning calorimetry. Further in-depth analysis based on free-energy decomposition indicates that destabilized binding can be attributed to electrostatic interaction induced by the chosen antigen module. These results provide molecular insights into the conformational stability of capsomeres and their abilities to be exploited for antigen presentation, and are expected to be beneficial for the biomolecular engineering of VLP vaccines.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Rotamer Dynamics: Analysis of Rotamers in Molecular Dynamics Simulations of Proteins

Given by χ torsional angles, rotamers describe the side-chain conformations of amino acid residues in a protein based on the rotational isomers (hence the word rotamer). Constructed rotamer libraries, based on either protein crystal structures or dynamics studies, are the tools for classifying rotamers (torsional angles) in a way that reflect their frequency in nature. Rotamer libraries are routinely used in structure modeling and evaluation. In this perspective article, we would like to encourage researchers to apply rotamer analyses beyond their traditional use. Molecular dynamics (MD) of proteins highlight the in silico behavior of molecules in solution and thus can identify favorable side-chain conformations. In this article, we used simple computational tools to study rotamer dynamics (RD) in MD simulations. First, we isolated each frame in the MD trajectories in separate Protein Data Bank files via the cpptraj module in AMBER. Then, we extracted torsional angles via the Bio3D module in R language. The classification of torsional angles was also done in R according to the penultimate rotamer library. RD analysis is useful for various applications such as protein folding, study of rotamer-rotamer relationship in protein-protein interaction, real-time correlation between secondary structures and rotamers, study of flexibility of side chains in binding site for molecular docking preparations, use of RD as guide in functional analysis and study of structural changes caused by mutations, providing parameters for improving coarse-grained MD accuracy and speed, and many others. Major challenges facing RD to emerge as a new scientific field involve the validation of results via easy, inexpensive wet-lab methods. This realm is yet to be explored.     

Molecular Dynamics Simulations for Metallic Nanosystems

Nanotechnology is a crucial field for future scientific development where many different disciplines meet. Computational modelization of nanometer-sized structures is a key issue in this development because (i) it allows a considerable saving of resources and costly experimental setups intended to fabricate nanometric test devices and (ii) nowadays the study of nanometric sized systems is feasible with thoroughly designed computational codes and relatively low cost computational resources. This article describes how molecular dynamics simulations, in combination with potentials obtained in the framework of the embedded atom method, are able to describe the properties of two systems of interest for the development of future nanoelectronic devices: metallic nanowires and metallic nanofilms. Our results show that nanowire stretching results in a series of well-defined geometric structures (shells) and that thin films experiment a crystallographic phase transition for a decreasing number of layers. In both cases, good agreement with experiments is found.     

Structural Basis of Type 2A von Willebrand Disease Investigated by Molecular Dynamics Simulations and Experiments

The hemostatic function of von Willebrand factor is downregulated by the metalloprotease ADAMTS13, which cleaves at a unique site normally buried in the A2 domain. Exposure of the proteolytic site is induced in the wild-type by shear stress as von Willebrand factor circulates in blood. Mutations in the A2 domain, which increase its susceptibility to cleavage, cause type 2A von Willebrand disease. In this study, molecular dynamics simulations suggest that the A2 domain unfolds under tensile force progressively through a series of steps. The simulation results also indicated that three type 2A mutations in the C-terminal half of the A2 domain, L1657I, I1628T and E1638K, destabilize the native state fold of the protein. Furthermore, all three type 2A mutations lowered in silico the tensile force necessary to undock the C-terminal helix 6 from the rest of the A2 domain, the first event in the unfolding pathway. The mutations F1520A, I1651A and A1661G were also predicted by simulations to destabilize the A2 domain and facilitate exposure of the cleavage site. Recombinant A2 domain proteins were expressed and cleavage assays were performed with the wild-type and single-point mutants. All three type 2A and two of the three predicted mutations exhibited increased rate of cleavage by ADAMTS13. These results confirm that destabilization of the helix 6 in the A2 domain facilitates exposure of the cleavage site and increases the rate of cleavage by ADAMTS13.     

Molecular Dynamics Simulations of Phospholipid Bilayers

Abstract

Molecular dynamics (MD) simulations at 37°C have been performed on three phospholipid bilayer systems composed of the lipids DLPE, DOPE, and DOPC. The model used included 24 explicit lipid molecules and explicit waters of solvation in the polar head group regions, together with constant-pressure periodic boundary conditions in three dimensions. Using this model, a MD simulation samples part of an infinite planar lipid bilayer. The lipid dynamics and packing behavior were characterized. Furthermore, using the results of the simulations, a number of diverse properties including bilayer structural parameters, hydrocarbon chain order parameters, dihedral conformations, electron density profile, hydration per lipid, and water distribution along the bilayer normal were calculated. Many of these properties are available for the three lipid systems chosen, making them well suited for evaluating the model and protocols used in these simulations by direct comparisons with experimental data. The calculated MD behavior, chain disorder, and lipid packing parameter, i.e. the ratio of the effective areas of hydrocarbon tails and head group per lipid (a_t/a_h), correctly predict the aggregation preferences of the three lipids observed experimentally at 37°C, namely: a gel bilayer for DLPE, a hexagonal tube for DOPE, and a liquid crystalline bilayer for DOPC. In addition, the model and conditions used in the MD simulations led to good agreement of the calculated properties of the bilayers with available experimental results, demonstrating the reliability of the simulations. The effects of the cis unsaturation in the hydrocarbon chains of DOPE and DOPC, compared to the fully saturated one in DLPE, as well as the effects of the different polar head groups of PC and PE with the same unsaturated chains on the lipid packing and bilayer structure have been investigated. The results of these studies indicate the ability of MD methods to provide molecular-level insights into the structure and dynamics of lipid assemblies.     

The Role of Molecular Dynamics Simulations for the Study of Slow Dynamics

Abstract

A two step strategy is proposed to study dynamical properties of a physical system much slower than the time scales accessible by molecular dynamics simulations. The strategy is applied to investigate the slow dynamics of supercooled liquids.     

Molecular Dynamics Simulations of NaCl-type Solid Solution Crystals: The First Application of Molecular Dynamics to Solid Solutions

Abstract

Solid solution crystals appear widely in the fields of earth sciences and inorganic material sciences. The physical properties of solid solutions may vary continuously with chemical composition. Sometimes, linear relationships of the properties with composition are assumed. However, this approximation is not always applicable (e.g., [1], [2], [3]). In order to elucidate the properties of solid solutions, studies on the relation between the macroscopic properties and the atomic configurations (microscopic property) in the crystal are desirable. One of the most effective approaches to the subject is molecular dynamics (MD). However, as far as the authors are aware there have been no molecular dynamics studies on solid solution crystals.     

Molecular Dynamics Simulations of Calcium-Free Calmodulin in Solution

Abstract

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmod- ulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 Å, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between ?2° and 108°. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34–45 Å range and its bend angle in the 10–100° range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.     

Modelling Diffusion in Zeolites by Molecular Dynamics Simulations

Abstract

The diffusion of molecules sorbed in zeolites is of growing interest for understanding the mechanisms of chemical processes with regard to selectivity and reactivity [1].

MD simulations give insight into physical systems on the molecular level allowing to study and visualize the motion of molecules even beyond the possibilities of experiments [2,3]. Single system parameters can easily be varied to study their influence, also those parameters that are fixed in reality (e.g., the size of particles). We present a cross section of our recent work to illustrate the capabilities of MD: The self diffusion coefficients (D) of a mixture of methane and xenon in silicalite show remarkable deviations from those of the pure species. This is shown and confirmed by PFG NMR experiments [4].

Simulating ethane in zeolite A the mechanism of diffusion has been studied. The effects of rotation on the diffusion lead to cases where D decreases with growing temperature [5].

The independence of self diffusion on lattice vibrations is proven even for zeolites with windows of guest particle size comparing simulations with rigid and vibrating zeolite lattice [6].     

Molecular Dynamics Simulations of Molecules in Uniform Flow

Flow at the molecular level induces shear-induced unfolding of single proteins and can drive their assembly, the mechanisms of which are not completely understood. To be able to analyze the role of flow on molecules, we present uniform-flow molecular dynamics simulations at atomic level. The pull module of the GRoningen MAchine for Chemical Simulations package was extended to be able to force-group atoms within a defined layer of the simulation box. Application of this external enforcement to explicit water molecules, together with the coupling to a thermostat, led to a uniform terminal velocity of the solvent water molecules. We monitored the density of the whole system to establish the conditions under which the simulated flow is well-behaved. A maximal velocity of 1.3 m/s can be generated if a pull slice of 8 nm is used, and high velocities would require larger pull slices to still maintain a stable density. As expected, the target velocity increases linearly with the total external force applied. Finally, we suggest an appropriate setup to stretch a protein by uniform flow, in which protein extensions depend on the flow conditions. Our implementation provides an efficient computational tool to investigate the effect of the flow at the molecular level.     

分子模拟在多黏菌素药理机制研究中的应用

多黏菌素是一种膜靶向的脂肽类抗生素,是临床上治疗革兰氏阴性多重耐药菌感染的最后一道防线。通过与脂多糖相互作用,多黏菌素破坏细菌外膜结构并导致细菌死亡。然而,受限于生物化学和结构生物学手段对细胞膜-药物相互作用的表征能力,目前对多黏菌素药理机制的认识还不充分,从而限制了新一代多黏菌素药物的设计和开发。为此,本文总结了近年来利用分子动力学方法对细胞膜系统与多黏菌素相互作用的研究进展,为深入理解多黏菌素药理机制与细胞膜系统的内在联系,加快新型抗生素药物研发提供新思路。     

The Molecular Mechanism Underlying Recruitment and Insertion of Lipid-Anchored LC3 Protein into Membranes

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys⁶⁵, Arg⁶⁸, and Arg⁶⁹, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.