模拟蛋白质折叠过程的新算法研究

蛋白质折叠过程模拟是当前蛋白质研究领域的一个难点问题。针对这一问题,提出了一个描述蛋白质折叠过程的算法-拟蛇算法,并且从分子振荡和分子动力学理论两个方面来证明该算法的核心函数是可行和正确的。经过实验总结出所有蛋白质空间结构都可以通过两种类型函数构造出来,提出了描述蛋白质折叠过程模型。与其它蛋白质折叠过程模拟算法的实验结果比较表明,拟蛇算法所构造的空间结构能量值最小、相似度最好。进而说明拟蛇算法和蛋白质折叠过程模型在描述蛋白质折叠过程方面具有明显优势。     

机械力作用下蛋白质结构和功能的转换

机械力作用下蛋白质结构和功能的转换涉及到纳米尺度范围内机械化学信号之间的转换。本文从以下几个方面对这一领域进行了综述：局部外力和几何感觉控制细胞功能：多结构域蛋白——纤连素在机械转化中的优点：将外力转化成生化信号;活细胞中细胞外基质中的纤连素在力诱导下的展开;纤连素Ⅲ型结构域通过序列变化调节机械稳定性：大肠杆菌Ⅰ型毛缘的展开结构有利于捕获键形成以及在药物和技术领域的应用。     

基于蛋白质受体的药物分子计算机辅助设计策略

药物分子计算机辅助设计是一种在计算机或者理论上通过构建具有一定潜在药理活性的新化学实体的分子模拟方法。近十几年来,高通量组学技术的快速发展为生物和化学药物分子设计提供了良好的数据支撑和研究契机。另外,现代社会对生物制药合理性以及作用机理理解的要求越来越高,行业普遍要求药物需要有高效、无毒或者低毒以及靶向性强等特点。随着越来越多与药物靶点相关的蛋白质结构通过实验方法解析出来,基于蛋白质受体的药物分子设计方法可行性进一步提高,其方法也变得越来越重要。基于蛋白质受体的药物分子设计方法,一般是以蛋白质以及配体的三维结构出发进行分析,这让药物分子先导物的发现更加理性化。随着相关实验数据的积累以及深度学习等算法的发展,从而可以进行更加科学的药物分子设计,这在一定程度上加快了新药研发的进程,并更有利于探索相应的分子机理。本文对基于蛋白质受体的药物分子设计方法的常用策略进行系统的回顾、总结和展望。     

基于计算机模拟的蛋白质分离纯化实验

利用数学建模的方法首次提出了衡量分离纯化效果优劣的定量指标——纯化效益;建立了便于应用推广的聚乙二醇(polyethyleneglycol,PEG)和硫酸铵沉淀分离纯化模型,并以海芋过氧化物酶分离纯化为例,对模型进行求解、应用和检验,求解最优实验试剂用量.     

蛋白质结构与功能研究中的分子模拟技术

分子模拟技术为蛋白质的研究提供了一种崭新的手段,在理论上解决了结构预测和功能分析以及蛋白质工程实施方面所面临的难题。它在蛋白质的结构预测和模建工作中占有举足轻重的地位,实现了生物技术与计算机技术的完美结合。本文简要阐述了该技术的基本步骤和工作原理,并以目前应用最广的生物大分子领域的商品化分子模拟软件Accelrys公司基于Linux系统开发的InsightII为例,介绍了相关程序模块的功能和作用,同时结合该技术在蛋白质的结构预测和模建、结构与功能关系分析、分子设计等过程中的开发与应用,加以具体说明和展望。     

蛋白质-蛋白质分子对接方法中分子柔性处理与近天然结构筛选的研究

蛋白质-蛋白质分子对接方法是研究蛋白质分子间相互作用与识别的重要理论方法。该方法主要涉及复合物结合模式的构象搜索和近天然结构的筛选两个问题。在构象搜索中,分子柔性的处理是重点也是难点,围绕这一问题,近年来提出了许多新的方法。针对近天然结构的筛选问题,目前主要采用三种解决策略:结合位点信息的利用、相似结构的聚类和打分函数对结构的评价。本文围绕以上问题,就国内外研究进展和本研究小组的工作作详细的综述,并对进一步的研究方向进行了展望。     

工业蛋白质构效关系的计算生物学解析

工业催化用酶已经成为现代生物制造技术的核心"芯片"。不断设计和研发新型高效的酶催化剂是发展工业生物技术的关键。工业催化剂创新设计的科学基础是对酶与底物的相互作用、结构与功能关系及其调控机制的深入剖析。随着生物信息学和智能计算技术的发展,可以通过计算的方法解析酶的催化反应机理,进而对其结构的特定区域进行理性重构,实现酶催化性能的定向设计与改造,促进其工业应用。聚焦工业酶结构-功能关系解析的计算模拟和理性设计,已成为工业酶高效创制改造不可或缺的关键技术。本文就各种计算方法和设计策略以及未来发展趋势进行简要介绍和讨论。     

双杂交和质谱技术在蛋白质-蛋白质相互作用研究中的应用

基因的功能是由蛋白质来执行的,而蛋白质要通过与其他生物分子相互作用来完成其各种生物功能。因此,如果能够快速做出蛋白质在不同时间、空间和不同环境中的相互作用图谱,就会帮助我们了解这些蛋白质的功能,进而了解许多生命活动的机制。目前,用于大规模研究蛋白质间相互作用的方法主要有酵母双杂交系统及其衍生系统、亲和纯化与质谱分析联用技术,前者用于研究蛋白分子间的两两相互作用,后者用于研究蛋白质复合物间的相互作用。本文主要阐述了酵母双杂交、细菌双杂交、哺乳动物细胞双杂交、亲和纯化与质谱联用技术在大规模蛋白质相互作用研究中的应用。     

核酸和蛋白质一级结构序列的计算机分析

本文介绍了计算机在核酸和蛋白质一级结构序列分析上的一些应用,包括序列的收集和贮存,两个或多个序列之间同源性的比较,用限制性内切酶找出酶切位点,找出DNA序列的开放式密码解读链和蛋白质序列倒翻成DMA序列的可能结果以及DNA和蛋白质序列的建立和应用等。     

跨膜蛋白CD3ε与磷脂酰肌醇二磷酸在脂筏域体系中蛋白质-脂质相互作用的粗粒化模拟研究

细胞膜局部区域可形成富含饱和脂质、胆固醇、鞘脂的脂筏域作为其信号转导调控平台。传统实验手段在研究脂筏及其功能时受到系统复杂度高及区域结构瞬时性强等制约。近年来,分子动力学模拟技术为细胞膜的组织原则提供了重要的理论支撑,从简单的单一组分模型到多组分系统转变,最终形成了越来越多的细胞膜仿真模型。其中,粗粒化模拟由于其简化模型,可大副拓展模拟体系的复杂程度与模拟时间,在细胞膜以及蛋白质-脂质相互作用相关研究中得到了广泛应用。本文采用MARTINI粗粒化力场模拟,构建了一种含有阴离子脂质磷脂酰肌醇二磷酸(phosphatidylinositol diphosphate, PIP2)的混合膜体系。模拟结果表明,该体系在适当温度及饱和度条件下,能自发分层形成脂筏域;膜厚度、膜组分分布、膜组分流动性等多种参数均表明,脂筏结构形成且符合其结构特征;少量PIP2添加不影响分层特性且PIP2对脂筏具有显著亲和性。此外,利用该模型以跨膜信号蛋白CD3ε为例研究了脂筏域体系中蛋白质-脂质相互作用。结果表明,PIP2-CD3ε胞内区相互作用可能是脂筏招募CD3ε的驱动力,且该过程可受钙离子调控。本工作体现了粗粒化模拟在仿真膜相关研究中的巨大优势及良好应用前景。     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Shear rate dependence of free energy barrier height for phenyl ring rotations in polystyrene based on non-equilibrium molecular dynamics simulations

Polystyrene properties are influenced by ring motions in side groups. The main chain conformation and interaction with the surroundings dominate the ring rotations. It is known that shear flow affects linear chain conformation and molecular distribution. However, shear-induced variations in the ring rotations have yet to be studied. This study presents a shear flow system of polystyrene via non-equilibrium molecular dynamics simulations. The free energy barrier of the phenyl ring rotations was obtained from the distribution of angle χ between the ring and main chain based on the Boltzmann distribution law. The results showed that the barrier height approaches a constant value at a shear rate less than 10¹⁰ s^? 1, but decreases with an increase in shear rate higher than 10^10.5 s^? 1. Furthermore, the radial distribution function and potential energies were compared. Remarkably, the shear flow reduced the bond vibrations of the phenyl rings, but increased the separation between intermolecular particles. Hence, a smaller cavity is necessary for the rings to rotate once but more volume is occupied by the rings. The smaller volume obtained via main chain motions needed to construct the cavity lowers the energy barrier height at shear rate higher than 10^10.5 s^? 1.     

Molecular simulations of phosphonium-based ionic liquid

Compared with imidazolium-based ionic liquids (ILs), phosphonium-based ILs have been proven to be more stable in thermodynamics and less expensive to manufacture. In this work, a kind of phosphonium-based IL, [PC₆C₆C₆C₁₄][Tf₂N], was studied under several conditions using molecular dynamics simulations based on both the all-atom force field (AAFF) and the united-atom force field. Liquid density was calculated to validate the force field. Compared with experimental data, good agreement was obtained for the simulated density based on the AAFF. Heat capacities at constant pressure were calculated at several temperatures, and good linear relationships were observed. Self-diffusion coefficients, viscosities and conductivities were also calculated to study the dynamics properties of this IL. The viscosity of this IL at 293 K was also compared with experimental data, and the error was in a reasonable range. In order to depict the microstructures of the IL, centre-of-mass and site-to-site radial distribution functions were employed. In addition, spatial distribution functions were investigated to present the more intuitive features.     

Princeton_TIGRESS: Protein geometry refinement using simulations and support vector machines

Protein structure refinement aims to perform a set of operations given a predicted structure to improve model quality and accuracy with respect to the native in a blind fashion. Despite the numerous computational approaches to the protein refinement problem reported in the previous three CASPs, an overwhelming majority of methods degrade models rather than improve them. We initially developed a method tested using blind predictions during CASP10 which was officially ranked in 5th place among all methods in the refinement category. Here, we present Princeton_TIGRESS, which when benchmarked on all CASP 7,8,9, and 10 refinement targets, simultaneously increased GDT_TS 76% of the time with an average improvement of 0.83 GDT_TS points per structure. The method was additionally benchmarked on models produced by top performing three‐dimensional structure prediction servers during CASP10. The robustness of the Princeton_TIGRESS protocol was also tested for different random seeds. We make the Princeton_TIGRESS refinement protocol freely available as a web server at http://atlas.princeton.edu/refinement . Using this protocol, one can consistently refine a prediction to help bridge the gap between a predicted structure and the actual native structure. Proteins 2014; 82:794–814. © 2013 Wiley Periodicals, Inc.     

Protein under pressure: molecular dynamics simulation of the arc repressor

Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.     

棉花纤维蛋白质3种提取及二维电泳方法的比较

高质量的蛋白样品制备是进行二维电泳的先决条件.棉花纤维中含有纤维素、多酚、多糖等严重干扰二维电泳的物质, 增加了蛋白提取和二维电泳的难度.分别采用3种提取植物组织蛋白的方法(水法、酚法和尿素法), 提取棉纤维总蛋白, 进而进行了二维电泳分析.在蛋白产量、蛋白纯度和电泳图谱等方面对3种方法进行了比较, 结果采用酚法提取的样品取得了较好的电泳图谱, 有望成为从棉纤维样品中提取总蛋白的可选方法.     

编码嗜麦芽黄单胞菌terminase-like蛋白基因的克隆与分析

根据Grover报道的XanthomonasmaltophiliaCG类受体的 342bp核酸序列设计的一特异引物P2和随机引物进行PCR扩增 ,将约 75 0bpPCR产物克隆到 pUCm T载体上 ,得到重组质粒 pUCm Ter。pUCm Ter上插入片段经M 13通用引物双向测序 ,其中ORF5 5 5核酸序列的 2 83～ 36 2bp部分与PseudomonasphageD3orf2基因的 1385～ 14 6 4bp部分有 86 %相同碱基。由ORF5 5 5编码 184aa蛋白序列的 1～ 16 5aa部分与PseudomonasphageD3orf2基因编码terminase的 2 2 9～ 393aa部分具有 6 6 %相同序列。因此 ,克隆到的ORF5 5 5核酸序列可能是编码嗜麦芽黄单胞菌terminase like蛋白的基因序列。     

Protein folding: Hypotheses and experiments

The current state of the problem of protein folding is reviewed with special attention to the novel molten globule state of the protein molecule, intermediate between the native and unfolded states. Experimental evidence on the existence of this state and its role in protein folding are compared with the sequential model of protein folding proposed by the author in 1972–1973.     

Free energy simulations for protein ligand binding and stability

Abstract

We summarize several computational techniques to determine relative free energies for condensed-phase systems. The focus is on practical considerations which are capable of making direct contact with experiments. Particular applications include the thermodynamic stability of apo- and holo-myoglobin, insulin dimerization free energy, ligand binding in lysozyme, and ligand diffusion in globular proteins. In addition to provide differential free energies between neighboring states, converged umbrella sampling simulations provide insight into migration barriers and ligand dissociation barriers and analysis of the trajectories yield additional insight into the structural dynamics of fundamental processes. Also, such simulations are useful tools to quantify relative stability changes for situations where experiments are difficult. This is illustrated for NO-bound myoglobin. For the dissociation of benzonitrile from lysozyme it is found that long umbrella sampling simulations are required to approximately converge the free energy profile. Then, however, the resulting differential free energy between the bound and unbound state is in good agreement with estimates from molecular mechanics with generalized Born surface area simulations. Furthermore, comparing the barrier height for ligand escape suggests that ligand dissociation contains a non-equilibrium component.