【作者】 刘宁宁；

【导师】 沈家骢； 张文科；

【作者基本信息】 吉林大学 ， 高分子化学与物理， 2011， 博士

【摘要】 核酸与蛋白质是组成生命的主要生物大分子,二者的相互作用构成了诸如生长、繁殖、遗传和代谢等生命现象的基础。从单分子水平上对它们的相互作用进行纳米探测,有利于人们更加深入地理解与调控这些重要的生理过程,是人们解开生命奥秘的关键所在。基于原子力显微镜的单分子力谱技术是一种非常有效的研究分子间/分子内相互作用的方法。本论文首先对单分子力谱的原理和它在生物学领域的应用进行了总结,然后通过对两个核酸-蛋白质体系的研究利用并发展了该力谱方法。这两个核酸-蛋白质体系分别为：(1)长链双螺旋DNA与SSB蛋白(2)烟草花叶病毒中基因组RNA与衣壳蛋白之间的相互作用。本论文的工作围绕以上两个体系并遵从由简入繁的原则展开：一、研究了双螺旋DNA在外力诱导下构象转变的本质以及单股DNA与SSB蛋白的相互作用。以长链双螺旋DNA作为探针,利用单分子力谱的方法并结合单股DNA结合蛋白(SSB)只与单股DNA结合而不与双螺旋DNA结合的特性,研究了双螺旋DNA在较低外力(65 pN左右)诱导下构象转变的本质。二、以更加复杂的完整烟草花叶病毒(TMV)为模型体系,研究基因组RNA与衣壳蛋白之间的相互作用。我们首次将RNA从TMV颗粒中牵拉出来,直接定量测得了基因组RNA与衣壳蛋白之间的结合强度,并证明衣壳蛋白保留在TMV蛋白外壳上而没有随RNA一同被牵拉出来。当外力被撤销后部分RNA能够重新组装回到TMV的蛋白外壳中。三、在上述工作基础上,更加深入地对RNA与衣壳蛋白解离的动力学过程进行了研究。通过测量在不用的pH值及外力加载速率下RNA与衣壳蛋白的断裂力,来描述RNA与衣壳蛋白断裂过程的能垒,获得其动力学信息,揭示RNA从蛋白外壳上解组装的机制。合强度,并证明了衣壳蛋白保留在TMV蛋白外壳中而没有随RNA一同被牵拉出来。当外力被撤销后部分RNA在位于5’端较远的、没有被破坏的RNA-蛋白外壳复合物的作用下,能够重新组装回到TMV的蛋白外壳中。我们还初步考察了环境因素,比如缓冲溶液中的EDTA浓度、pH值以及外力加载速率对RNA与衣壳蛋白相互作用的影响。通过此研究我们将AFM单分子力谱方法拓展到相对复杂生物体系的核酸与蛋白质相互作用的研究中,同时有望将该技术应用到病毒感染机理的研究。在本论文第四章中,仍以烟草花叶病毒为研究模型,更加深入、系统地对RNA与衣壳蛋白解离的动力学过程进行了研究。通过测量在不用加载速率下RNA与衣壳蛋白的断裂力,发现该断裂力是具有加载速率依赖性的。而且,Bell-Evans模型可以很好地拟合我们的实验数据,并给出了RNA与衣壳蛋白的断裂过程的动力学参数并且描述了断裂的能垒。在pH值为4.7的条件下RNA与衣壳蛋白的解离过程由一个能垒控制；在pH值为7.0的条件下断裂过程由两个能垒控制,增加的一个能垒来源于病毒颗粒内壁上无序的突环结构对RNA解离的阻碍作用。更多还原

【Abstract】 Nucleic acids and proteins are two important biomacromolecules that compose the life, and the interactions between them are the base of many biological phenomena, such as growth, propagation, inheritance, metabolism, and so on. The nano-mechanical detection of nucleic acid-protein interactions at single-molecule level will deepen our understanding and eventually gain controls on these biological processes. This is also the key to explore the mysteries of life. Atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) is a very effective technique for the investigation of inter- or intramolecular interactions.In chapter 1, the basic principle of SMFS is introduced in detail, including the analysis of a force curve, the criteria for single chain stretching and dynamic force spectroscopy (DFS). Some recent progresses in SMFS study of biological systems have also been summarized. In the later chapters, we investigated two nucleic acid and protein systems from relatively simple to complex one by using SMFS. These two systems are long double stranded DNA (dsDNA) and single-stranded DNA (ssDNA) binding protein (SSB), together with genetic RNA of tobacco mosaic virus (TMV) and its coat proteins, respectively.In chapter 2, we have revealed the mechanism of force-induced conformation transition of dsDNA and the interactions between (single stranded DNA) ssDNA and SSB proteins. Taking advantage of the character that SSB proteins interact with ssDNA specifically but not with dsDNA, we used a long dsDNA as a probe to investigate the nature of force-induced conformation transition of dsDNA. Our results indicate that dsDNA is partially melted into ssDNA during the overstretching transition (i.e. dsDNA exist as a mixture of the dsDNA and molten ssDNA) at the mechanical force of about 65 pN, and the SSB proteins are able to capture the transient ssDNA fragments slowing down the rehybridization process, causing the hysteresis between stretching and relaxation traces. After relaxation, the SSB proteins can be removed from the ssDNA fragments, and the dsDNA recovers its B-form conformation. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. The study indicates that electrostatic interaction, intercalating interaction and hydrophobic interaction are the main driving forces for the formation of SSB-ssDNA complexes.In chapter 3, we took a more complicated system, an intact tobacco mosaic virus (TMV), as a model to reveal the interactions between genetic RNA and coat proteins. For the first time, by using an AFM tip, we have pulled the genetic RNA step by step out of the helical groove formed by its protein coat, producing a sawtooth-like plateau, from which the quantitative unbinding force between the RNA and coat proteins is obtained. We have proved that the coat proteins stayed in the protein coat but not being pulled out together with RNA. When the external force is released, the detached RNA fragment can find its way back to the helical protein coat with the help of intact RNA-protein complexes located in the deeper part (i.e., away from the 5’end) of the TMV particle. We have also studied the effects of other factors such as EDTA concentration, pH value and loading rate on the interactions between RNA and coat proteins. The present study extends the force spectroscopy technique to the study of nucleic acid-protein interactions in more complicated biological systems. And the method established here may open a new door toward investigations of the mechanism of virus infection.In chapter 4, we still took tobacco mosaic virus as a model system to investigate the dynamic process of RNA-coat protein interactions in more detail. We explored the dependence of unbinding forces of RNA-coat protein complexes on the loading rate, that is, the rate at which the force is applied to the binding. The experimental data can be fitted well by the Bell-Evans model, which enables us to determine the energy barrier width (xp), off-rate constant (koff), binding lifetime (τ) and to describe the energy landscape of RNA-coat protein interactions. The results show that at pH 4.7, the unbinding process is only dominated by one energy barrier stabilizing RNA-coat protein complexes, while at pH 7.0, the unbinding process is dominated by two energy barrier, the extra energy landscape comes from the disordered loop of polypeptide in the wall of the inner channel of TMV.更多还原