蛋白质-表面活性剂组装结构的分子模拟

采用Langevin分子动力学方法模拟β模型蛋白与表面活性剂在溶液中形成的各种组装结构,考察了表面活性剂疏水性强度与浓度对蛋白质分子构象的影响.结果显示：弱疏水性表面活性剂可以在蛋白质表面自组装形成限制性空间,使被包覆的蛋白质的立体结构更为稳定;强疏水性的表面活性剂则可以与蛋白质疏水核心区的疏水基团形成复合物,而使蛋白质的立体结构被破坏,即蛋白质发生去折叠.上述模拟可再现相关实验结果,其展现的蛋白质结构转换微观图景对于表面活性剂的分子设计及其应用于生物加工过程具有指导作用.     

液固层析辅助蛋白质从变性态恢复到活性态

层析是化工中分离纯化一些价格昂贵的精细化工产品的常用手段,而液固层析因其温和、快捷、高效等优点已经成为蛋白质纯化过程中必不可少的工具.近年来层析技术的一个拓展是用于辅助蛋白质从变性态恢复到活性态,即所谓的复性,取得了令人瞩目的进展.液固层析辅助蛋白质复性可在高蛋白浓度下操作,适用于大规模生产,同时还能使目标蛋白得到部分纯化.对近年来发展的利用液固层析辅助蛋白质复性的方法做了归纳,包括凝胶过滤层析、离子交换层析、疏水相互作用层析、亲和层析和固定化的折叠催化剂及人工分子伴侣辅助蛋白质复性的方法,并对各自的优缺点和适用范围进行了比较分析.     

离子液体表面活性剂的合成与应用(Ⅺ)——与生物活性分子的相互作用

从3方面综述了离子液体表面活性剂与生物活性分子的相互作用。亲油性的离子液体表面活性剂可与β-环糊精形成1:1,1:2或2:1型包合物,而亲水性的离子液体表面活性剂不能与β-环糊精形成包合物,这些包合物的形成影响了表面活性剂的cmc和在界面的吸附。离子液体表面活性剂能够与牛血清蛋白、β-酪蛋白、溶菌酶等蛋白质相互作用,影响蛋白质的分子构型以及在油/水界面的吸附状态。离子液体表面活性剂与DNA的相互作用主要来源于静电吸引和疏水作用。     

热变性对蛋白质结构及泡沫行为的影响

以牛血清白蛋白(BSA)和鸡蛋白蛋白(albumin)为模型蛋白,研究了热变性对蛋白质起泡性能和分子结构的影响. 结果表明,热变性后的BSA起泡能力下降,泡沫稳定性有增强的趋势. 热变性使BSA分子表面巯基含量下降,分子之间发生缔合,表面疏水性下降. 而albumin在热变性后的起泡能力及泡沫稳定性都大大提高,热变性使albumin分子展开,表面巯基含量增加. 实验证明,蛋白质分子的表面疏水性是决定其起泡能力的重要因素之一,蛋白质分子之间的相互作用对泡沫的稳定性有很大的影响.     

复配表面活性剂对油水界面行为和性质影响的模拟研究

含复配表面活性剂的油田废水是一种多组分复杂体系,研究其中的分子作用关系有助于后续废水处理方案的确定。采用分子动力学(MD)模拟方法建立界面模型,通过定义表面活性剂的关键扭转点及相应的分子夹角、定义协同作用能,结合界面处的密度分布函数等性能模拟和界面张力测试结果,多角度分析两类阴阳离子表面活性剂复配对油水界面特性的影响。结果表明,与含单组分表面活性剂的油水体系相比,复配表面活性剂的相反电荷极性头基间静电吸引作用提高了油水界面稳定性;相较于十二烷基磺酸钠/十六烷基三甲基溴化铵(SLS/CTAB)复配体系,十二烷基硫酸钠/十六烷基三甲基溴化铵(SDS/CTAB)中分子间的协同作用可更好地提高体系的稳定性;当复配表面活性剂的配比为8/10~12/6时的油/水界面稳定效果较优、12/6时稳定性最好。研究结果可为石油开采及油水分离方案的确定提供依据。     

复配表面活性剂对油水界面行为和性质影响的模拟研究

含复配表面活性剂的油田废水是一种多组分复杂体系,研究其中的分子作用关系有助于后续废水处理方案的确定。采用分子动力学(MD)模拟方法建立界面模型,通过定义表面活性剂的关键扭转点及相应的分子夹角、定义协同作用能,结合界面处的密度分布函数等性能模拟和界面张力测试结果,多角度分析两类阴阳离子表面活性剂复配对油水界面特性的影响。结果表明,与含单组分表面活性剂的油水体系相比,复配表面活性剂的相反电荷极性头基间静电吸引作用提高了油水界面稳定性;相较于十二烷基磺酸钠/十六烷基三甲基溴化铵(SLS/CTAB)复配体系,十二烷基硫酸钠/十六烷基三甲基溴化铵(SDS/CTAB)中分子间的协同作用可更好地提高体系的稳定性;当复配表面活性剂的配比为8/10~12/6时的油/水界面稳定效果较优、12/6时稳定性最好。研究结果可为石油开采及油水分离方案的确定提供依据。     

高分子抑制蛋白质聚集的动态Monte Carlo模拟

抑制聚集是蛋白质产品下游加工特别是制剂过程中的重要问题。本文采用动态Monte Carlo方法和二维晶格HP蛋白质模型,通过建立高分子-蛋白质复合物微观结构和蛋白质构象概率分布来研究高分子对蛋白质聚集行为的影响。结果表明,高分子的疏水性、分子量及其浓度对于蛋白质的聚集行为有显著的影响。当其疏水性适宜时,高分子可富集在蛋白表面疏水位点,强化蛋白质分子在水溶液中的分散,从而抑制聚集。高分子还可缠绕在蛋白质分子表面形成限制性空间从而稳定蛋白质的天然结构。     

蛋白质在逆胶束中增溶的分子热力学模型

为建立文题所述模型，以微乳液的稳定性理论为基础，综合分析了表面活性剂分子在油－水界上的缔合作用、界面膜弯曲时产生的附加能量、各种类型的混合熵以及表面活性剂分子和蛋白质分子的静电作用。所得模型正确地反映了含蛋白质的逆胶束系统系统稳定的原因，较好地描述了蛋白质转移后胶束大小的变化以及蛋白质转移后胶束大小的变化以及蛋白质转移率随电解质浓度变化的特殊的实验规律。     

利用体积排阻色谱法进行蛋白质折叠

以溶菌酶为模拟体系对体积排阻色谱法进行蛋白质折叠过程实验研究 .圆二色性光谱法分析结果证实了复性溶菌酶与天然溶菌酶的二级结构一致性 ;复性溶菌酶与天然溶菌酶色谱保留体积的差异揭示出折叠过程中无活性蛋白质聚集体的存在及其向复性蛋白质转化的机制 ;不同初始浓度的复性实验证实了蛋白质聚集体的存在及其与变性蛋白质初始浓度的关系 ;采用短色谱柱的折叠分离实验结果表明蛋白质折叠是一个快速过程 ;不同尿素浓度下的折叠分离实验结果表明尿素在SEC法中具有非常重要的作用 .与稀释复性法的对比实验表明 :体积排阻色谱法具有稀释倍数小、复性产品活性收率高、复性蛋白质浓度高等优点 .     

分子动力学模拟二硫键对大豆11S球蛋白结构及表面活性的影响

采用分子动力学模拟分析了二硫键对天然和还原状态下大豆11S球蛋白结构及其表面活性的影响。模拟结果表明,二硫键断裂后蛋白质分子稳定性降低,蛋白质分子的构象和二级结构发生变化;二硫键全部断裂会增大蛋白质分子的柔性,有利于表面活性的提高。而断开Cys88-298和Cys12-45、Cys88-29之间的二硫键会增大蛋白质疏水区域的溶剂可及表面,说明这两种断裂方式会增大蛋白质分子在界面的吸附量,有利于界面张力的降低,可以有效提高蛋白质的表面活性。     

Is Protein Folding a Thermodynamically Unfavorable,Active, Energy-Dependent Process?

The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy G. We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that ΔG of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.     

TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding

The various effects of native protein folding on the stability and folding rate of intrinsically disordered proteins (IDPs) in crowded intracellular environments are important in biomedicine. Although most studies on protein folding have been conducted in vitro, providing valuable insights, studies on protein folding in crowded intracellular environments are scarce. This study aimed to explore the effects of intracellular molecular crowding on the folding of mutant transactivator HIV-1 Tat based on intracellular interactions, including TAR RNA, as proof of the previously reported chaperna-RNA concept. Considering that the Tat–TAR RNA motif binds RNA, we assessed the po tential function of TAR RNA as a chaperna for the refolding of R52Tat, a mutant in which the argi nine (R) residues at R52 have been replaced with alanine (A) by site-directed mutagenesis. We mon itored Tat-EGFP and Tat folding in HeLa cells via time-lapse fluorescence microscopy and biolayer interferometry using EGFP fusion as an indicator for folding status. These results show that the refolding of R52A Tat was stimulated well at a 0.3 μM TAR RNA concentration; wild-type Tat refolding was essentially abolished because of a reduction in the affinity for TAR RNA at that con centration. The folding and refolding of R52Tat were mainly promoted upon stimulation with TAR RNA. Our findings provide novel insights into the therapeutic potential of chaperna-mediated fold ing through the examination of as-yet-unexplored RNA-mediated protein folding as well as viral genetic variants that modulate viral evolutionary linkages for viral diseases inside a crowded intra cellular environment.     

Refolding of Lysozyme in Glycerol as Studied by Fast Scanning Calorimetry

The folding of lysozyme in glycerol was monitored by the fast scanning calorimetry technique. Application of a temperature–time profile with an isothermal segment for refolding allowed assessment of the state of the non-equilibrium protein ensemble and gave information on the kinetics of folding. We found that the non-equilibrium protein ensemble mainly contains a mixture of unfolded and folded protein forms and partially folded intermediates, and enthalpic barriers control the kinetics of the process. Lysozyme folding in glycerol follows the same or similar triangular mechanism described in the literature for folding in water. The unfolding enthalpy of the intermediate must be no lower than 70% of the folded form, while the activation barrier for the unfolding of the intermediate (ca. 140 kJ/mol) is about 100 kJ/mol lower than that of the folded form (ca. 240–260 kJ/mol).     

Folding of Trp-cage Mini Protein Using Temperature and Biasing Potential Replica��Exchange Molecular Dynamics Simulations

The folding process of the 20 residue Trp-cage mini-protein was investigated using standard temperature replica exchange molecular dynamics (T-RexMD) simulation and a biasing potential RexMD (BP-RexMD) method. In contrast to several conventional molecular dynamics simulations, both RexMD methods sampled conformations close to the native structure after 10–20 ns simulation time as the dominant conformational states. In contrast, to T-RexMD involving 16 replicas the BP-RexMD method achieved very similar sampling results with only five replicas. The result indicates that the BP-RexMD method is well suited to study folding processes of proteins at a significantly smaller computational cost, compared to T-RexMD. Both RexMD methods sampled not only similar final states but also agreed on the sampling of intermediate conformations during Trp-cage folding. The analysis of the sampled potential energy contributions indicated that Trp-cage folding is favored by both van der Waals and to a lesser degree electrostatic contributions. Folding does not introduce any significant sterical strain as reflected by similar energy distributions of bonded energy terms (bond length, bond angle and dihedral angle) of folded and unfolded Trp-cage structures.     

Impact of the C‐terminal Disulfide Bond on the Folding and Stability of Onconase

The two homologous proteins ribonuclease A and onconase fold through conserved initial contacts but differ significantly in their thermodynamic stability. A disulfide bond is located in the folding initiation site of onconase (the C‐terminal part of the protein molecule) that is missing in ribonuclease A, whereas the other three disulfide bonds of onconase are conserved in ribonuclease A. Consequently, the deletion of this C‐terminal disulfide bond (C87–C104) allows the impact of the contacts in this region on the folding of onconase to be studied. We found the C87A/C104A‐onconase variant to be less active and less stable than the wild‐type protein, whereas the tertiary structure, which was determined by both X‐ray crystallography and NMR spectroscopy, was only marginally affected. The folding kinetics of the variant, however, were found to be changed considerably in comparison to wild‐type onconase. Proton exchange experiments in combination with two‐dimensional NMR spectroscopy revealed differences in the native‐state dynamics of the two proteins in the folding initiation site, which are held responsible for the changed folding mechanism. Likewise, the molecular dynamics simulation of the unfolding reaction indicated disparities for both proteins. Our results show that the high stability of onconase is based on the efficient stabilization of the folding initiation site by the C‐terminal disulfide bond. The formation of the on‐pathway intermediate, which is detectable during the folding of the wild‐type protein and promotes the fast and efficient refolding reaction, requires the presence of this covalent bond.     

海藻糖对变性溶菌酶的促进复性作用及动力学

研究了海藻糖促进变性还原溶菌酶的复性作用,考察了不同盐酸胍浓度下海藻糖浓度对复性收率的影响,并利用表观竞争反应动力学模型分析了海藻糖辅助溶菌酶的复性动力学特性.结果表明,在海藻糖存在下,聚集体生成速率常数k_A明显减小,但复性速率常数k_N变化不大;随海藻糖浓度的增大,k_A先降低后升高,在合适浓度时k_A出现最小值,由于此时k_N/k_A出现最大值,因此复性收率最大.说明海藻糖的主要作用是有效抑制蛋白质分子间聚集,因此在一定范围内提高其浓度可促进蛋白质复性,从而提高复性收率.     

复杂液氮洗装置的工艺模拟与改造研究

某厂液氮洗装置工艺复杂,能量集成度较高,本文应用Aspen Plus流程模拟软件对该装置设计工况进行模拟,并对热力学模型进行对比和评价,认为RK-ASPEN热力学模型能够准确表述其热力学行为;其次通过模拟原料气条件变化,而且无弛放气来源的情况,提出了合理可行的改造方案.     

Refolding of proteins in a CSTR

A model to predict refolding of proteins in a continuous stirred tank reactor (CSTR) was developed and compared to a batch refolding process with simple dilution of the protein in a stirred tank reactor. For experimental verification of the model a continuous refolding of a model protein (α-lactalbumin) was performed in a CSTR. The refolding process of denatured and fully reduced α-lactalbumin could be accurately predicted by a set of differential equations assuming a first order reaction rate for folding and a second order reaction rate for aggregation. The system composed of a CSTR with an additional diafiltration circuit for removal of denaturing agents from the feed solution and to maintain constant refolding conditions. Based on the folding kinetic the dynamic behavior of such a continuous refolding reactor was simulated under different operating conditions. It was shown that the refolding efficiency was higher compared to batch dilution under certain conditions, namely high residence times. The yield of refolded protein could further increased by recycling the outlet stream containing unfolded protein to the reactor entrance.