| 题名 | A型γ-氨基丁酸受体与药物作用的分子模拟及其亚基的克隆表达 |
| 作者 | 次素琴 |
| 学位类别 | 博士 |
| 答辩日期 | 2008-02-01 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 苏志国 |
| 关键词 | GABAA受体结构 分子模拟 体外表达 定点突变 光谱 |
| 其他题名 | Molecular Simulation on the Interaction of γ-Aminobutyric Acid Type A Receptor with Drug, and Cloning and Expression of its Subunit |
| 学位专业 | 化学生物学★ |
| 中文摘要 | A型γ-氨基丁酸（aminobutyric acid，GABA）受体是哺乳动物中枢神经系统内最主要的抑制性神经递质受体，其分子结构上存在众多药物的作用位点，是目前医药和农药领域研究的热点。但因膜蛋白的纯化技术的落后，GABAA受体晶体结构领域的研究一直未有突破。本论文通过计算机模拟和分子生物学实验手段，对GABAA受体结构及其与配体、激动剂、非竞争性拮抗剂的作用方式进行了探索研究，主要工作内容如下： 1. 采用MODELLER软件构建了哺乳动物大脑内GABAA受体α1β2γ2、α1β3γ2和α6β3γ2 3种亚型以及α1γ2、α2γ2、α3γ2、α5γ2 4种二聚体模型。在此基础上，在已有突变实验结论的指导下，通过对接（docking）方法探索了GABA和地西泮（diazepam）与α1β2γ2亚型的可能作用方式、GABA与α1β3γ2和α6β3γ2两种亚型亲合性的差异以及唑吡坦（zolpidem）药物对α1γ2、α2γ2、α3γ2、α5γ2 4种二聚体模型的选择性。 2. 采用同源模建技术，以烟碱型乙酰胆碱（nicotinic acetylcholine）受体的电镜结构为模板，成功构建了β3同源5聚体通道模型，并以其为靶点，通过对接方法探索了两类共6种氟虫腈（Fipronil）相关杂环化合物与其可能的作用方式。该5聚体模型的建立为解决目前昆虫GABA受体组成尚未明了，无法作为药物靶点进行研究的问题提供了一个有效的解决方案。同时，化合物半抑制浓度（IC50）与对接能量的线性关系分析为以GABA受体为靶点的新化合物的活性预报提供了可能。 3. 在计算机模拟的基础上，采用分子生物学实验手段在先于全部结构解决之前优先研究了GABAA受体的部分结构信息。将哺乳动物GABAA受体的α1亚基和β3亚基基因通过RT-PCR扩增，克隆至原核表达载体pET-30a中，含α1亚基的重组质粒在E.coli BL21（DE3）细胞中以包涵体方式获得了高效表达，通过肽质量指纹图谱（peptide mass fingerprint, PMF）分析，鉴定目的基因在体外得到了正确表达。 4. 以前期的分子模拟结论为指导，将α1亚基中位于配体，激动剂及拮抗剂作用位点内的7个关键氨基酸残基进行了定点突变，测序结果表明，突变位点正确，且无移码突变。突变质粒转化E.coli BL21（DE3）细胞，均获得了高效表达。通过包涵体洗涤、变性、Ni2+亲合层析纯化，稀释复性获得纯化的重组表达蛋白。应用圆二色谱测定纯化的原始及突变蛋白的二级结构，比较突变位点对蛋白原始结构的影响，同时结合远紫外光谱分析蛋白突变前后对药物结合力的差异。 本文将计算机模拟与分子生物学实验相结合，对GABAA受体的部分结构及其与药物的作用模式进行了初步探索，可望对以GABAA受体为靶点的新药开发提供参考。 |
| 英文摘要 | γ-Aminobutyric acid type A receptor (GABAA receptor) is the major inhibitory neurotransmitter receptor in the central nervous system of mammalian brain. It is a great hotspot in the medicinal and insecticidal areas because there are many drug binding sites on the GABAA receptor. However, our understanding of the molecular details of this receptor has been limited by a lack of high-resolution structural information. In the present work, we study the information about 3-dimensional structure of GABAA receptor by using the technology of computational simulation and molecular cloning. Molecular docking approach is employed to analyze the binding-mode of ligand, agonist and the non-competitive antagonist with GABAA receptor. The contents of the study are as follows: 1. The three-dimensional structure of the GABAA receptor that included the ligand/agonist binding site was constructed and validated by molecular modeling technology. Moreover, according to the results of mutational experiments, the putative binding-mode of GABA and diazepam with α1β2γ2 subtype were investigated by means of docking studies. In addition, the differences of ligand-binding domains of α1β3γ2 and α6β3γ2 subtypes and the selectivity of zolpidem to those receptors containing α1, α2, α3 or α5 subunits were also studied. 2. Using the cyro-electron microscopy structure of nicotinic acetylcholine as the template, the three-dimensional model of the β3-homopentamer was developed by homology modeling. The interaction of this model with two classes of fipronil related heterocyclic compounds were investigated by docking studies. The subunit composition of native insect receptor is not clear in the current study，so we proposed that the β3 homooligomer can be used as an alternative model to study the interaction modes of noncompetitive antagonists with the insect GABA receptor. Moreover, correlation between the binding energies calculated by AutoDock and experimentally determined semi-inhibitory concentration (IC50) values could be used to predict the activities of new compounds which targeted the insect GABA receptor. 3. Based on the results of molecular modeling, the strategy of resolving individual domain structures pior to an attempt to reconstitute the overall structure was proposed. In this regard, the segment of α1 and β3 subunit was amplicated by RT-PCR. By using prokaryotic expression vector pET-30a, α1 subunit fragment was overexpressed in Escherichia coli by a fusion protein with His-tag located in N-terminal. The analysis of peptide mass fingerprint (PMF) showed the recombinant protein was correctly expressed in vitro. 4. According to the result of molecular modeling, seven point mutations were made on the residues in the putative ligand/agonist binding pocket and the second transmembrane domain. DNA sequencing confirmed all sequences were right and no frameshift mutation was found. The mutated vectors were also expressed with high level in E. coli BL21 (DE3) cells. The purified protein was gained through denature, washing, Ni2+ affinity chromatography purification and the refolding of inclusion bodies. The secondary structure of the wide-type and the mutated protein were performed by CD spectroscopy. In addition, UV spectrum was used to analysis the binding differences of the drug to the original and the mutated protein. In the present work, the combination of computational simulation and molecular biology was used to investigate GABAA receptor structure and the drug interaction mode. The research results will give the references to the design of new medicine and pesticide which target GABAA receptor. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 154 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1122]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 次素琴. A型γ-氨基丁酸受体与药物作用的分子模拟及其亚基的克隆表达[D]. 过程工程研究所. 中国科学院过程工程研究所. 2008. |
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