化学小分子诱导的蛋白质活性中心周围微环境的变化

发布时间：2017-12-30 17:49

本文关键词：化学小分子诱导的蛋白质活性中心周围微环境的变化　出处：《南京师范大学》2016年博士论文　论文类型：学位论文

【摘要】：蛋白质是生命的物质基础,一切生命活动离不开蛋白质。蛋白质在生命体内的活性和功能取决于其结构和空间构象。由于蛋白质的空间结构主要依靠氢键、范德华力、静电力等非共价作用的共同平衡而维持,但这些作用力较弱且易受温度、酸碱度、压力、离子强度、外源性分子作用等因素影响而发生改变,导致蛋白质构象和功能发生改变,甚至导致某些疾病的产生。与蛋白质结合的某些化学小分子,包括药物分子对蛋白质维持结构和功能的稳定性具有极其重要的作用。因此,研究化学小分子与蛋白质相互作用机理和规律,有助于深入了解蛋白质构象与功能的关系,解释外源性分子在生物体内的结合、运输过程,也有助于在分子水平上解释某些疾病的致病机制,为某些复杂疾病的基础研究和防治等提供重要的理论指导。本论文研究了化学小分子与蛋白质的相互作用机理,及作用后蛋白质活性中心周围微环境的变化。以葡萄糖氧化酶、人血清白蛋白和肌红蛋白为蛋白质分子模型,利用电化学方法、多种光谱法如：紫外可见吸收光谱法、荧光光谱法(包括荧光淬灭法、同步荧光光谱法、荧光共振能量转移法、时间分辨荧光光谱法)、红外光谱法、圆二色谱法,以及分子模拟计算等,研究了溶液中有机小分子(以硫酸胍为例)、抗肿瘤药物小分子(以5-氟尿嘧啶为例)、光敏剂(以酞菁锌衍生物为例)和不同pH条件分别对以上蛋白质进行诱导后蛋白质发生的结构变化机理,及诱导后蛋白质活性中心周围微环境的变化。本论文的主要内容及结果如下：1.研究了有机小分子(以Gdm+为代表)诱导蛋白质(以葡萄糖氧化酶(GOx)为模型)结构及其活性中心周围微环境变化的机制。利用电化学方法,研究了不同浓度Gdm+诱导后对GOx的电催化活性与其构象的关系。结合紫外-可见吸收光谱法、红外光谱法、圆二色谱法及分子模拟计算等方法深入研究了Gdm+诱导的GOx结构与其催化性能的关系。结果表明,Gdm+与GOx作用时能进入其疏水腔使GOx发生去折叠过程,GOx的a-螺旋结构含量减少,户折叠结构含量增加；分子模拟研究发现Gdm+与GOx的氧化还原活性中心FAD作用,使FAD分子本身的电子性质、与周围氨基酸残基的氢键等发生了明显改变,这是导致GOx的二级、三级结构变化的决定性因素,也进一步使GOx对葡萄糖的催化氧化活性明显降低。2.研究了抗肿瘤药物小分子(以5-氟尿嘧啶(FU)为代表)与人血清白蛋白(HSA)的相互作用机理及FU-HSA结合位点微环境的变化。研究结果表明,FU分子能够进入HSA分子中Trp 214残基附近的ⅡA域,并与Trp 214和Lys 199残基形成氢键,从而导致Trp 214残基发生了静态荧光淬灭。分子对接和热力学参数分析的结果表明,HSA与FU的结合主要由范德华力和氢键驱动形成了FU-HSA复合物,从而导致蛋白质微环境比天然结构更加疏水。此外,HSA与低浓度(150 μmol/L)时的FU作用使α-螺旋结构含量增加,而与高浓度(150 μmol/L)的FU作用时则使α-螺旋结构含量减少,这可能是由于蛋白质在此作用过程中发生了一定程度的扭转导致。3.研究了光敏剂(以酞菁锌衍生物(ZnPcs:ZnPc1、ZnPc2、ZnPc3)为代表)人血清白蛋白(HSA)的相互作用及ⅡA域微环境变化的机制。研究结果表明,三种ZnPcs衍生物都进入了HSA分子中Trp 214残基附近的ⅡA域内,且分别与HSA形成了ZnPcs衍生物-HSA复合物,使Trp214周围环境更亲水；作用过程中Trp214残基发生了静态荧光淬灭,并伴随一定程度的动态淬灭过程；HSA与三种ZnPcs衍生物的结合主要通过静电引力和疏水作用力形成了ZnPcs衍生物-HSA复合物,此外,与ZnPcl和ZnPc2相比,由于ZnPc3静电势较高,ZnPc3衍生物与HSA作用后使HSA的静电荷分布变化最大,且ZnPc3使Trp214周围残基氢键的变化也最大,说明ZnPcs三种衍生物中,ZnPc3与HSA的作用最强。4.研究了不同pH环境诱导下肌红蛋白(Mb)结构及血红素周围微环境变化的机理,建立了一种检测溶液中含血红素蛋白结构及其血红素周围微环境变化的简单方法。基于循环伏安法测量Mb在不同pH缓冲溶液中的电子转移信号能够灵敏地反映出Mb分子的活性中心血红素基团周围结构的微妙变化。在酸性和碱性溶液中,由于血红素中Fe-His 93配位键断裂,血红素基团从Mb的疏水腔中分离而暴露于溶剂中,使Mb在溶液中的电子转移信号增强。结合紫外可见光谱法、拉曼光谱法、CD光谱法和分子模拟计算研究了不同pH条件下Mb结构和活性中心血红素基团周围微环境的变化,结论与电化学方法研究结论一致,说明电化学方法可用于检测氧化还原蛋白Mb在溶液中的结构变化,与其他方法相比,电化学方法具有反应灵敏、操作简便、成本低廉等优点,并且该方法可以反映溶液中蛋白质结构变化的动力学过程。
[Abstract]:Protein is the material basis of life, all life activities cannot do without protein. The activity and function of proteins in vivo depends on its structure and conformation. The protein structure mainly depends on the hydrogen bond, van Edward, common balance electric non covalent interactions and maintain, but these forces are weak and vulnerable temperature, pressure, pH, ionic strength, exogenous molecules and other factors change, leading to protein conformational and functional changes, and even cause some diseases. And the protein binding of certain chemical molecules, including plays an extremely important role in the stability of drug molecules to maintain protein structure and function. Therefore, study on chemical molecules and protein interaction mechanism and law, contribute to a deeper understanding of the relationship between protein conformation and function, explain the exogenous molecules in biological In combination, the transportation process, but also help in the molecular level to explain pathogenesis of some diseases, and provide important theoretical guidance for some complex diseases research and prevention. The interaction mechanism of the study of chemical molecules and proteins, and the role of protein changes around the active center of the micro environment. With glucose oxidase, human serum albumin and myoglobin protein molecular model, using electrochemical method, spectroscopic methods such as UV Vis absorption spectroscopy, fluorescence spectroscopy (including fluorescence quenching method, synchronous fluorescence spectroscopy, fluorescence resonance energy transfer assay, time-resolved fluorescence spectroscopy, infrared spectroscopy, circular) two chromatography and molecular simulation, studied organic molecules in solution (with guanidine sulfate as an example), small molecule drugs (5- fluorouracil for example), photosensitizer (in zinc phthalocyanine Derivatives as an example) and different pH conditions were changed after the induction of protein structure mechanism of protein, and protein induced changes around the active center of the microenvironment. The main contents and results of this thesis are as follows: 1. of small organic molecules (represented by Gdm+) induced protein (with glucose oxidase (GOx as the model mechanism of surrounding structures)) and the active center of micro environmental changes. Using the electrochemical method, the relationship between the electrocatalytic activity of GOx and the conformation of the effects of different concentrations of Gdm+. After induction with UV Vis absorption spectroscopy, infrared spectroscopy, the relationship between round two chromatography and molecular simulation calculation method research the structure and catalytic performance of GOx induced by Gdm+. The results showed that Gdm+ and GOx can enter the hydrophobic cavity of the GOx to reduce the content of the folding process, a- helix structure of GOx, the user node folding The content increased; molecular simulation study found that the oxidation of Gdm+ and GOx reduction activity center FAD, the electronic properties of FAD molecule itself, and the surrounding amino acid residues hydrogen changed obviously, which is the two level to GOx three level, the decisive factor of structural changes, but also further the catalytic oxidation activity of GOx on glucose significantly decreased the.2. of small molecule drugs (5- fluorouracil (FU) as the representative) and human serum albumin (HSA) change site micro environment with interaction mechanism and FU-HSA. The results show that the FU molecule can enter the domain near the molecule of HSA II A Trp 214 residues, and the formation of hydrogen bonds between Trp 214 and Lys 199 residues, resulting in Trp 214 residue static fluorescence quenching. Analysis of molecular docking and thermodynamic parameters showed that the combination of HSA and FU mainly by van Edward driving force and hydrogen bond formation The FU-HSA complex, which leads to the protein microenvironment more hydrophobic than natural structure. In addition, HSA with low concentration (150 mol/L) FU is the alpha helix content increased, and the high concentration (150 mol/L) FU is the alpha helix structure which can reduce the content. This is due to the protein effect in the process of the reverse occurs to a certain extent on the.3. (zinc phthalocyanine derivatives photosensitizer leads to (ZnPcs:ZnPc1, ZnPc2, ZnPc3) as the representative) human serum albumin (HSA) mechanism of interaction and domain II A microenvironment. The results showed that three kinds of ZnPcs derivatives into the II A domain near HSA molecule Trp 214 residues, and HSA respectively with the formation of ZnPcs derivatives of -HSA complexes, the Trp214 environment more hydrophilic; role in the process of Trp214 residue static fluorescence quenching and dynamic quenching, with a certain degree of death; The combination of HSA and three kinds of ZnPcs derivatives mainly by electrostatic force and hydrophobic force to form derivatives of ZnPcs -HSA complex, in addition, compared with ZnPcl and ZnPc2 ZnPc3, due to the electrostatic potential is high, ZnPc3 derivatives with HSA. After the HSA static charge distribution and the change of the biggest change, ZnPc3 Trp214 is also the largest hydrogen bonding residues around ZnPcs, three kinds of derivatives, ZnPc3 and HSA.4. had the strongest effect on different pH environment under the induction of myoglobin (Mb) mechanism and structure around the heme microenvironment change, a simple method of environment around the heme heme containing changes in protein structure and a detection solution. The cyclic voltammetry in electronic measurement Mb different pH buffer solution transfer signal can sensitively reflect subtle changes in structure around the active center of the heme group based on Mb molecule. In acidic and alkaline solution, The heme Fe-His 93 ligand bond cleavage, heme group separation and exposed to the solvent from the hydrophobic cavity of the Mb, the Mb signal of electron transfer in solution increased. Combined with ultraviolet visible spectroscopy, Raman spectroscopy, study the changes around different pH conditions Mb structure and active center of micro heme group the calculation of CD spectroscopy and molecular modeling, the research conclusion and electrochemical methods are consistent, the electrochemical method can be used to detect structural changes of redox protein Mb in solution, compared with other methods, the electrochemical method has sensitive reaction, simple operation, low cost, and the method can reflect the dynamic change process of protein structure in the solution.

【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

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