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用单分子磁镊研究大肠杆菌单链结合蛋白的结合性质
中文摘要

单链结合蛋白(Single-strand DNA binding protein,SSB)可以以很高的亲和力结合单链DNA(ssDNA),并可以与许多DNA代谢相关蛋白发生相互作用,从而起到保护ssDNA产物不被降解,并介导代谢相关蛋白进入相应位置进行反应的功能。大肠杆菌单链结合蛋白(E-coli SSB)是单链结合蛋白中具有代表性的一种,它是一种同源四聚体,每个亚基都可以和ssDNA产生高度亲和力并结合。大肠杆菌SSB存在三种稳定的结合模式,分别结合65 nt ssDNA、56 nt ssDNA和35 nt ssDNA,分别在不同盐离子浓度下存在。许多研究者对大肠杆菌SSB这一现象进行了大量的研究,然而,其反应的具体过程以及相关反应系数仍然没有被全部揭示。 因此,我们利用单分子动力学的方法,结合单分子磁镊进行拉力实验,观察SSB在拉力下的反应现象,发现在某个临界力下,SSB会发生跳变式结合/解离现象。我们对这个现象进行了长时间测量,得出其反应系数以及相应的自由能参数。我们通过对自由能的修正,还原了拉力为零时大肠杆菌SSB的结合过程,发现它是一个由快速地结合和缓慢地缠绕相组合的过程,表现形式代表了结合过程强度的不均匀性。通过分析,我们认为这是由于SSB上电荷密度不均匀导致的反应现象,可能与生理过程中的调节有关。 我们在不同的NaCl浓度下进行了一系列类似SSB的拉力测试。我们发现,在不同的盐离子浓度下,都会出现类似的跳变式结合/解离现象。然而随着盐离子浓度的变化,SSB的反应系数以及发生跳变的临界力也会发生相应变化,在特定的盐离子浓度下,SSB的反应参数会出现明显的跳变,显示出不同结合模式的特征。我们测定了反应自由能的变化规律,发现SSB与ssDNA的结合呈现出一级相变的规律,自由能在临界点存在拐点。我们还可以通过得到的参数计算出结合中的熵变以及解离的离子数变化,发现不同结合模式之间SSB结合的熵变以及解离离子数存在明显区别,并且发现SSB的不同结合模式是由于蛋白自身在不同的盐溶液中不同性质导致的。我们的结果比较完整的展示了盐离子浓度对SSB结合的影响。 我们在不同的盐离子种类下进行了类似的拉力研究,发现当盐离子种类不同时,结合的过程及特征也不同。我们认为,这个现象进一步说明SSB的表面电荷特征会极大的影响其与ssDNA的结合。 通过我们的实验,在单分子层面揭示了单链结合蛋白与单链DNA的相互作用机理,也为类似的大分子相互作用提供了研究思路。 关键词:单链结合蛋白,磁镊,单分子动力学,反应系数,自由能

英文摘要

Single-stranded binding protein (SSB) can bind ssDNA in high affinity. It can also interact with different kinds of proteins, which attend in DNA metabolism. SSB can protect ssDNA from degradation and lead DNA metabolic protein to their working site. E-coli SSB is a representational SSB among all kinds of SSB. It is a homotetramer that each subunit can binds ssDNA in high affinity. E-coli SSB has three stable binding modes, which binds 65 nt ssDNA, 56 nt ssDNA and 35 nt ssDNA respectively under different salt concentration. Many researchers tried to study this phenomenon. However, the reaction process and reaction rate of SSB still remain unclear. We use single molecule dynamics combine with single molecule magnetic tweezers pulling experiment to observe and analyse the reaction between SSB and ssDNA. And we find that under a critical force, a combine/dissociation jump happens. We measure this reaction and get the reaction rate and free energy parameters. We calibrate the free energy to exhibit SSB binding process under zero force. We find that SSB binding process consists of rapid combination and slow wrapping, whose binding force are different. We assume that different electronic density among SSB surface leads to this phenomenon. Maybe it is related to the metabolize regulation. We repeat this experiment under different NaCl concentrations. We find that the combine/dissociation jump happens under different NaCl concentrations. SSB reaction rate and critical force change under different NaCl concentrations. Under special NaCl concentration, the reaction rate changes in large scale, which represents different binding mode. We measure the change of reaction free energy. The result shows that the binding process of SSB and ssDNA has a characteristic like first order phase transition, and a inflexion point can be found under some critical NaCl concentration. We can also calculate the binding entropy change and released ion number, and find their change regularity. We conjecture that different characteristics of SSB under different salt solution lead to different SSB binding mode. Our results exhibit the influence of ion concentration to SSB binding process completely. We do a pulling experiment under different salt specis, and find that the SSB binding process and characteristics are different from those under NaCl solution. We consider that this phenomenon can provide proof about the influence of SSB surface electronic density to the binding process of SSB/ssDNA. Our results indicate the difference of SSB binding processes under different salt conditions, and make the details of different SSB binding modes clearer. Key Words: single-stranded DNA binding protein, magnetic tweezers, single molecule dynamics, reaction rate, free energy

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