【作者】 梁涛；

【导师】 王吉华； 王传奎；

【作者基本信息】 山东师范大学 ， 原子与分子物理， 2016， 硕士

【摘要】 固有无序蛋白P53是人体中与肿瘤癌症密切相关的蛋白,在细胞信号转导网络中起着关键作用。CBP(CREB binding protein)是P53的一个调节蛋白,通过它固有的乙酰化作用,在细胞的转录修饰机制中起到支架作用。P53和CBP是多位点结合,其中NCBD(CBP蛋白碳端区域)与TAD(P53蛋白N端转录激活域)之间的结合,对P53与DNA形成四聚体起到关键的作用。实验发现单体的TAD是完全无序的,而单体的NCBD是部分无序的,但是它们结合形成的复合物是有序结构。这表明结合过程是一个结构特性改变的过程。研究无序蛋白P53与CBP的作用模式,不仅有助于了解它们结合时构象的变化,还有助于了解无序蛋白P53的调节机制。分子动力学模拟是目前蛋白质研究的有力工具。本论文运用分子动力学模拟的方法,研究固有无序蛋白P53与蛋白CBP的相互作用,通过计算其结合自由能、分析三级结构变化等对P53对蛋白CBP的构象变化和稳定性的影响。具体结果如下:1.本文对P53蛋白N端转录激活域TAD单体(由49个残基组成)、CBP蛋白碳端区域NCBD单体(包含59个残基)和二者结合的复合体(包含108个残基)分别进行了长时间分子动力学模拟,研究了结合前后各种结构参数的变化情况。为从原子层次揭示无序蛋白P53与CBP的作用模式,进而认识P53的调节机制有重要意义。2.研究结果表明,在结合前,TAD单体和NCBD单体都是无序的,二者均表现出很大的柔性,只包含有稀疏的结构,不能形成稳定的结构,但是,当两个蛋白结合以后,TAD和NCBD形成复合体的自由能要比单体TAD与NCBD的自由能低,这说明结合后系统更加稳定,而且形成复合体后,柔性变小,形成螺旋的概率变大。3.研究结果进一步表明,在单体TAD和NCBD结合成复合体时,二级结构也发生了很大变化。当TAD和NCBD结合以后,TAD新生了成两个螺旋结构,而且最初两个螺旋之间的距离变大,无规则卷曲结构增加;同时,NCBD中螺旋α1与螺旋α3的距离开始变大,而螺旋α1与螺旋α2的距离开始变小;而且,部分二级结构中螺旋和无规则卷曲出现的概率变大,从而增加了系统结合后的稳定性;这也进一步说明了无序蛋白在结合过程中更容易形成螺旋和无规则卷曲这一特点。本论文分为四部分内容。第一章为绪论,介绍了固有无序蛋白的结构特点以及生理功能,其中CBP蛋白和P53蛋白都是固有无序蛋白,在细胞转录以及细胞信号转导网络中起着重要的作用。然后重点介绍了CBP蛋白和P53蛋白的结构组成、相关疾病以及CBP蛋白NCBD域和P53蛋白TAD域。第二章为分子动力学模拟,介绍了分子动力学模拟的相关知识,分子动力学模拟的原理、分子力场和分析方法、使用的软件GROMACS以及分子动力学模拟的前景。第三章使用分子动力学模拟研究P53与CBP相互作用的结果进行了分析,通过研究apo-TAD,apo-NCBD和复合体的结合分析了CBP蛋白对P53蛋白构象稳定性的影响。第四章总结了本研究的主要结论,并展望了未来的发展。更多还原

【Abstract】 Intrinsically disordered protein P53, which has close relation with tumor, plays a very important role in cell signal transduction network. CBP(CREB binding protein) is a regulation protein of P53, which plays role of scaffolding by its inherent acetylation in cell transcription modification mechanism. There are many binding sites between CBP and P53, especially the binding site between the NCBD(C-terminal domain of CBP protein) and the TAD(N-terminal transcription activation domain of P53 protein). It was found that the TAD monomer is complete disordered, and the monomer NCBD is partly disordered, but their complex is orderly structure. This shows that the binging process is a process of changing their structures. The research not only helps to understand the change of the conformations when they are combined, but also helps to understand the regulatory mechanism of the disordered protein P53.Molecular dynamics(MD) simulation is a powerful tool used to study proteins. In this paper, the MD simulations were used to study interaction of the intrinsically disorderly protein P53 and CBP protein. The MD trajectories were analyzed to provide stability and conformation information by calculating the free energy, the tertiary structure and so on of the monomer TAD, the NCBD and their complex. The main results are as follows:1. TAD monomer which is N-terminal transcription activation domain of P53(consists of 49 residues), NCBD monomer of CBP protein(included 59 residues) and their complex(included 108 residues) are completed for long time molecular dynamics simulations, respectively. Based on the MD simulations, all kinds of the structural parameters are analyzed, which is important to understand the regulation mechanism of P53 and to reveal the interaction mode between the intrinsically disordered protein P53 and CBP at atomic level.2. The research results show that TAD monomer and NCBD monomer is disordered before the combination, which is very flexible and only contains some sparse structure and can’t form stable structure. However, when TAD and NCBD are combined with each other, the free energy of the complex is much lower than the free energy of the single monomers TAD with NCBD, showing that the combination complex is more stable, lower flexible and bigger probability forming the secondary structure helix.3. The results further show that the secondary structures change much when the monomers NCBD and TAD combined into the complex. The two new helix structures were found, and the distance between the original two helixes become larger, and the random curls increased too. In the MD simulation of the complex, the distance between helix α1 and helix of α3 of NCBD increased, and the distance between helixes α1 and α2 become smaller than those in the monomers NCBD and TAD, and the secondary structure helixes and random coil appear bigger probability. The results show that the complex is more stable.The paper is divided into four parts. The first chapter is introduction, introducing the intrinsically disorderly protein and their physiological function, and the CBP protein and P53 protein are disordered, then we introduced the structure domains NCBD, TAD and the relation with human diseases. The second chapter introduced the MD simulation, including the MD simulation, the experience force field, the principle of MD simulation, the analysis method, GROMACS software and the MD developing. In the third chapter, MD simulation was used to study the apo-TAD, apo-NCBD and the complex. The fourth chapter is the conclusion and prospect.更多还原