两类蛋白质与核酸相互作用的计算分析研究
文献类型：学位论文
| 作者 | 高跃东 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 黄京飞 |
| 关键词 | 蛋白质核酸相互作用 NAD+依赖的DNA连接酶 非键作用能 结合自由能 |
| 其他题名 | Investigating the interaction of protein-nucleic acid by computational analysis of two complex structures |
| 学位专业 | 动物学 |
| 中文摘要 | 蛋白质和核酸是生命体中最为重要的两类生物大分子。细胞各种重要的生物学过程（信号的转导、细胞对外界环境及内环境变化的反应等）是通过生物体内蛋白质与其他物质的相互作用为纽带来实现的。蛋白质与核酸的相互作用是分子生物学研究的中心问题之一。虽然蛋白质与核酸相互作用的研究已经取得了一定的研究成果，但仍有许多问题有待解决。进一步研究核酸被蛋白质识别的机制及其相互作用需要生物学、化学、物理学、计算机学等多学科的协作。在本工作中，我们通过对两类关键蛋白质（NAD+依赖的DNA连接酶和PTB蛋白）与核酸的相互作用的研究，揭示了这两类蛋白质与核酸的识别特点，以及他们相互结合过程在序列和结构上的规律，讨论了这些特点、规律和蛋白质功能的关系，进而加深了我们对蛋白质与核酸的相互作用的理解。 DNA连接酶属于共价的核苷酸转移酶超家族，在DNA的复制，重组和修复中起了关键作用，是一类古老而重要的酶。DNA连接酶利用NAD+或ATP中的能量催化DNA中相邻的5′磷酸基与3′羟基间形成磷酸二酯键，使DNA切口封合，连接DNA片段。目前DNA连接酶与nicked DNA的识别机制仍然没有被完全认识。在本工作中，我们首次在NAD+依赖的DNA连接酶中发现了该类酶特有的三个motif。通过在残基水平计算复合物的非键结合能，显示出这些motifs位于与nicked DNA结合的区域，其中的R136（motif A）、R200（motif B）、R333（motif C）高度保守且具有强相互作用。分析还显示，先前定义的该家族的五个特征motifs中，仅有三个具有较强的非键作用。进一步分析了其他DNA连接酶的复合物非键相互作用能量后，我们提出了DNA连接酶识别和结合DNA的假说，解释了DNA连接酶利用不同拓扑结构识别nicked DNA的现象。 本文的另一部分工作是分析了PTB蛋白与RNA结合的亲合性以及研究了其与RNA中CU富集区的特异识别机制。PTB（Polypyrimidine tract binding protein）是一类真核生物中普遍存在的RNA结合蛋白，其中的主要功能之一是作为抑制因子参与了mRNA的可变剪切。PTB蛋白特异识别CU富集的RNA区，其识别机制善待阐明。我们发现了PTB蛋白中新的结合区，即在PTB蛋白beta结合面的beta4上的丝氨酸及临近的极性残基特异结合RNA的胞嘧啶C。我们利用基于MM/PBSA方法计算结合自由能的protocol进行PTB蛋白与RNA的结合紧密程度的分析，计算并比较了特异结合胞嘧啶C的氨基酸突变前后的结合自由能，突变体的结合自由能大幅升高，表明突变体结合RNA较之天然结构极不稳定，即beta4结合区对RNA结合至关重要。动力学模拟显示，特异结合的残基和胞嘧啶C能稳定的形成氢键，显示出特异结合的特点。PTB蛋白的Beta4上的这一结合区是新发现的结合位点，其具有对RNA的强亲合力和特异识别RNA中胞嘧啶的特性。 在本研究中，围绕DS和PP软件平台进行的计算模块开发主要采用了三种策略：一是通过整合Perl程序到DS的Scripts菜单栏中，在DS可视化界面下进行操作。二是通过Pipeline Pilot整合componet形成新的protocol。三是从命令行调用DS函数集进行批量计算。我们发展的自动化批量计算残基水平非键相互作用能的protocol和基于MM/PBSA的结合自由能计算的protocol已经应用在我们对蛋白和核酸相互作用的研究中。 |
| 英文摘要 | Protein and nucleic acid are important cellular macromolecules. All cellular processes(signal transduction, cell response to extracellular and intracellular environmental change)depend on precise interactions mediated by proteins. Protein-nucleic acid interaction is central question in molecular biology. Despite progress in macromolecules interaction research, the description of the interactome represents one of key challenges remaining for structural biology. Cooperation in multi-knowledge based on biology, chemistry, physics and computer are considerable for further research. We focus on two crucial protein in organisms: NAD+ dependent DNA ligase and PTB (polypyrimidine-tract binding protein) protein and elucidate the interaction of these complex. We reveal the characteristic on the protein-nucleic acid recognition and the sequence-structure-function relationships in the two interaction system. DNA ligases belong to the covalent nucleotidyltransferases. DNA ligases catalyze the joining of nicked DNA, which is necessary in DNA replication and repair pathways where the re-synthesis of DNA is required. DNA ligase is an ancestral and crucial enzyme. Most organisms use ATP powered DNA ligases, but eubacteria appear to be uniquely use ligases driven by NAD+. Hitherto, it is not clear understand how the ligases recognize DNA. In the former part of this work, we report for the first time, three novel motifs unique in NAD+ dependent DNA ligase are not defined in previous studies. A highly automated calculation for non-bonded energy approach is created. It calculates the non-bonded interaction energies between the residues in the motifs and bases in DNA. The result shows that the major contribution for the recognition of ligase-DNA is the residues which in the motifs: Arginine136 (motif A), Arginine200 (motif B), Arginine333 (motif C). Only three motifs previous definition show high interaction. A DNA nick recognition model for the DNA ligases was proposed and can explain DNA ligase biding in all available complexs. Another part of this work is to study binding affinity and molecular basis for CU-rich RNA recognition in PTB protein. PTB（Polypyrimidine tract binding protein） is observed in eukaryote ubiquitously. Function of PTB protein is its role as a regulator of alternative splicing. PTB protein preferentially binds pyrimidine-tract containing CU-rich tracts, the mechanism is unknown. Our analysis shows that serine and vicinal plar residues which located beta4-strand of RRM domain play crucial role to binding RNA. It is novel site.The customize Pipeline Pilot Protocols has been set up, which can calculate the binding free energy of complexes based on MM/PBSA method. We create mutant by conseved residues in beta4 and calculated each binding free energy. Compared to the wt PTB protein binding free energy, we find that, mutant increased the energy largely. Dynamic ananlysis show that hydrogen bond is crucial for specific bind to C pyrimidin. In addition, we develop module based on DS. Three strategys was considered. Firstly, we integrate Perl code into DS menue, and run in DS windows. Secondly, we create protocol by Pipeline Pilot and import to Discovery Studio. Thirdly, accessible the DS programming interface by Perl code and run from the command line without open the DS client windows. The extension module applies to our analysis of protein- nucleic acid complex. |
| 语种 | 中文 |
| 公开日期 | 2013-04-24 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7403]  |
| 专题 | 昆明动物研究所_结构生物信息学 |
| 推荐引用方式 GB/T 7714 | 高跃东. 两类蛋白质与核酸相互作用的计算分析研究[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
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