【作者】 吴韬；

【导师】 郑鹏；

【作者基本信息】 南京大学 ， 化学， 2019， 硕士

【摘要】 单分子力谱技术能够在溶液体系内捕获单个分子,并对分子施加外力操纵其形变同时测量分子的稳定性,甚至断裂其中的化学键得到键强,从而被广泛的应用于化学和生物学的研究中。以原子力显微镜为基础的单分子力谱可以测量皮牛顿级别的微小作用力和记录纳米级的分子长度变化,从而被广泛的应用于生物大分子稳定性的研究中,例如蛋白质的机械稳定性、受体-配体相互作用以及蛋白中化学键的强度。但是精确识别和检测单个分子的行为仍然较为困难。近年来随着嵌合多聚蛋白构建技术、蛋白质位点特异性固定化技术以及受体-配体互作技术的发展,它们陆续被应用于单分子力谱样品的制备和操作中。这些技术的应用使得单个分子的拉伸解折叠事件被更加高效的获得和确认,单分子力谱技术也在近十年内得到了飞速发展和应用。本论文中我们首先提出了一种利用OaAEP1蛋白连接酶(Oldenlandia affinis asparaginyl endopeptidase)高效快速制备单分子力谱所需的多聚蛋白样品的方法。然后同样采用该蛋白酶,结合受体-配体互作技术,实现了多聚蛋白分子的表面固定化。最后,我们联合使用OaAEP1蛋白连接酶和TEV蛋白切除酶(tobacco etch virus protease)实现了蛋白模块的逐个精准添加,可以较好的控制所需多聚蛋白的聚合度和种类。第二章中我们利用OaAEP1酶将蛋白单体N端的“GL”和C端的“NGL”残基交联形成肽键得到多聚蛋白分子。同时我们研究了在不同环境下(酶用量、反应时间、反应体系pH、金属离子)酶活性的变化,实验表明OaAEP1酶具有用量少、反应快、适用pH广泛以及金属离子耐受度高等特点。我们成功地利用该方法将泛素蛋白(ubiquitin,Ub)和一种金属蛋白红素氧还蛋白(rubredoxin,RD)制备为多聚蛋白分子,应用于单分子力谱测量。实验结果表明该连接没有对蛋白的机械稳定性造成影响,多聚的效果也满足单分子力谱实验的需求。作为一种不需要引入半胱氨酸的蛋白交联方法,OaAEP1的酶交联方法更适合多聚金属蛋白质的构建。第三章中我们利用OaAEP1酶分别将之前制备的多聚蛋白Ub和RD固定到基板上,并在分子中插入较长的多肽片段ELP(elastin-like polypeptide)作为分隔,最后利用受体-配体相互作用实现了位点特异性固定化的单分子力谱实验。实验表明这种利用OaAEP1酶催化连接的方法能够同时完成蛋白的多聚与固定化两种功能,并且高效的完成单分子的捕获和测量,比以往非特异的技术效率提高近50倍。最后一章中我们将OaAEP1蛋白连接酶与TEV蛋白酶联合使用,在TEV酶切位点“ENLYFQG”序列后面加入一个亮氨酸,TEV酶消化位点位于谷氨酰胺和甘氨酸之间,因此切割后产生新的“GL”可以被OaAEP1酶识别。构建Cohesin与Ub/RD的融合蛋白,并在连接片段中间加入“tev-L”这样的切割位点,这样在单分子实验中我们可以逐个增加蛋白模块,并控制添加蛋白模块的数目和不同模块之间的交替连接。我们用SDS-PAGE验证了 TEV酶切后产生的“GL”可以被用于连接,且TEV酶消化反应十分完全。通过调节连接反应中Coh-tev-L-Ub-NGL和GL-Ub两种底物的比例,可以使GL-Ub反应的效率得到很大的提高。两种高效的反应为基板上的序列控制提供了基础。最后进行了控制Ub1～Ub5、RD1～RD5以及Ub和RD两种模块交替添加的单分子实验,通过得到的力曲线统计计算受控蛋白的比例,实验中增加一个模块进行实验后能够达到要求的力谱曲线概率～80%,从而证明我们可以较为精确地得到聚合度在五以内的多聚蛋白分子。本论文中我们主要利用了OaAEP1酶和TEV酶设计了一系列方案,实现多聚蛋白质分子的快速制备、蛋白的位点特异性固定化和有序控制的多聚蛋白分子制备。这些方法极大地提高了单分子力谱实验的准确度和效率,有望在今后的单分子研究中得到广泛的应用。更多还原

【Abstract】 Single-molecule force spectroscopy technique can capture single molecule in solution and apply external force to the molecule to measure the mechanical stability and chemical bonds strength by manipulating it.Therefore,it has been widely used in chemical and biological research.Single-molecule force spectroscopy based on atomic force microscopy is widely used in the study of the stability of biological macromolecules because it can measure the small force of pico-newton and record the length changed at the nanometer level,such as the mechanical stability of proteins,the binding strength of receptor-ligand interactions,and the strength of chemical bonds.However,it is difficult to precisely identify and detect the behavior of individual molecule.In recent years,chimeric polyproteins construction technology,site-specific immobilization technology,and receptor-ligand interaction technology have been applied to sample preparation and manipulation of single-molecular force spectroscopy.We can identify the stretching and unfolding of individual molecule,and singlemolecule force spectroscopy technology has also been widely used.In this thesis,we first propose to use OaAEP1 ligase to construct polyprotein samples efficiently and rapidly for single-molecule force spectroscopy experiments.Then the prepared polyproteins are immobilized on the substrate by OaAEP1 ligase and the receptor-ligand interaction technology.Finally,we combine OaAEP1 ligase with TEV protease to control the addition of protein modules one by one,which can better control the polymerization degree and variety of polyproteins.In the second chapter,we used the OaAEP1 ligase to construct polyproteins by crosslinking the "GL" at the N-terminus and the "NGL" residue at the C-terminus of protein monomers to form a peptide bond.We studied the enzyme activities in different environments(such as enzyme dosage,reaction time,pH,metal ions)in the first place.The experiment results showed that a bit of OaAEP1 could rapidly construct protein polymer in a wide pH range and can undertake multiple metal ions.We successfully used this method to prepare poly-ubiquitin and poly-rubredoxin for single-molecule force spectroscopy experiments.The experiment results showed that the connection does not affect the mechanical stability of proteins,and the multi-polymerization effect also meets the requirement of single-molecule force spectroscopy experiments.The OaAEP1 cross-link method is more suitable for the construction of poly-metalloprotein because it does not require the introduction of cysteine.In chapter three,we successfully used the OaAEP1 enzyme to immobilize the poly Ub and RD proteins on the substrate and then used the receptor-ligand interaction in single-molecule force spectroscopy experiments to stretch the immobilized polyproteins.We also inserted longer polypeptide fragment ELP as a partition in the molecule.The experiment results showed that OaAEP1 ligase can simultaneous perform the functions of multimerization and immobilization of proteins without affecting the mechanical stability of the protein.We could capture and measure single molecules more efficiently,which was nearly 50 times higher than non-specific technology.In the last chapter,we combined OaAEP1 ligase with TEV protease to control the covalent linkage of the monomer.We added a leucine after the TEV cleavage site of"ENLYFQG" amino acid sequence.The TEV protease proteolysis takes place between glutamine and glycine.When we add a leucine after the glycine,TEV protease digestion can create a new site "GL" that can be recognized by the OaAEP1.The fusion protein of Cohesin and Ub/RD was constructed,and a cleavage site such as "tev-L" was added in the middle of the ligation fragment.In the single-molecule force spectroscopy experiment,we can increase the protein module one by one and control the number and sequence of linked protein modules.We confirmed the ligation of "GL" produced by TEV digestion by SDS-PAGE,and calculated the TEV digestion reaction is quite complete.By adjusting the ratio of the two reactants of Coh-tev-L-Ub-NGL and GLUb in the ligation reaction,the efficiency of the GL-Ub reaction can be greatly improved.High-efficiency of OaAEP1 ligation and TEV hydrolysis make it possible to control protein modules on the substrate.Finally,single-molecule experiments were carried out by controlling the polymerization degree of Ub and RD modules.We calculated the reaction yield by counting the unfolding force peaks of particular protein domain in the force-extension curves.In each experiment,the probability of the addition of one more module is～80%,which proved that we can get the desired polyprotein molecules with the polymerization degree within five.In this paper,we used OaAEP1 ligase and TEV protease to design a series of schemes to rapidly prepare polyprotein samples,immobilize the polyproteins on the substrate,and control the sequence of prepared polyproteins.This method could greatly improve the accuracy and efficiency of single-molecule force spectroscopy experiments and is expected to be widely used in the future.更多还原