【作者】 李娟；

【导师】 田长麟；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 蛋白质是生物体行使功能的最重要的生物大分子之一,研究蛋白质在生理过程中的作用机制能够帮助我们更好的了解生命的意义。大部分蛋白质功能的实现依赖着其动态变化,核磁共振由于能够研究生物大分子在不同时间尺度上的运动特性而成为研究蛋白质动力学和作用机制的重要工具。本文应用生物化学方法分析了植物激素脱落酸(ABA)在拟南芥中的水溶性受体家族RCAR1/PYR/PYL中的成员PYL10对其下游信号因子2C类蛋白磷酸酶PP2C的作用机制。纠正了前人因偶然因素得出“PYL10是一类不依赖于ABA的受体”的结论。本文还进一步应用液体核磁共振弛豫测量和约化谱密度函数分析方法揭示了PYL10在ABA结合前后的动态特性,表明PYL10在结合ABA之后使得其结构熵和蛋白质柔性的增加。同时,本文还介绍了发展和应用无标记(Label-Free)质谱定量检测的方法研究大肠杆菌酪氨酸激酶C端酶活结构域(ETK-CTD)在磷酸化过程中的关键位点Tyr574的磷酸化水平的动态变化的工作,及简要介绍了植物内向整流钾离子通道KAT1的初步电生理研究。第一章先简要介绍了ABA受体的发现和分类。接着对水溶性的ABA受体RCAR1/PYR/PYL家族介导的ABA信号通路的主要内容及其在植物体中的重要功能、RCARl/PYR/PYL家族ABA受体结合ABA时的结构变化和发挥作用的机制进行了重点介绍。第二章主要介绍了基于液体核磁共振自旋弛豫的蛋白质动力学分析方法,并相关研究表明热力学原理和动力学的结合能够更好地解释蛋白质-配体或者蛋白质-蛋白质相互作用机制。第三章介绍了采用钼酸盐与磷酸根的显色反应检测PYL10在体外对PP2C(HAB1)磷酸酶活性的抑制,发现了与之前报道的结果(ABA非依赖性)相反的结果,即ABA依赖性,且这种依赖性会受到外源蛋白牛血清蛋白(BSA)的影响。生化分析结果表明PYL10和BSA不能形成稳定的复合物。第四章针对BSA不是通过与PYL10直接相互作用影响其功能,可能是使PYL10蛋白的动力学发生了变化而对功能产生影响。我们对自由状态的PYL10(apo-PYL10)和结合有ABA的PYL10(ABA/PYL10)进行了液体核磁共振主链归属、弛豫数据的采集和约化谱密度函数分析以及ABA滴定实验,结果表明PYL10对PP2C的磷酸酶活性抑制作用是通过ABA介导的主链柔性以及构象熵的增加实现的。第五章我们分别采用Label-Free质谱相对定量和Western-blot定性分析了ETK-CTD在磷酸化过程中自磷酸化位点Tyr574磷酸化水平的变化和蛋白质总磷酸化水平变化。质谱结果表明,ETK-CTD的Tyr574位点的磷酸化很快达到一个较低的稳定水平(1%)；而Western-blot结果显示,在Tyr574达到这个稳定水平后蛋白总体磷酸化依然升高最后保持稳定,表明仅仅一个很低水平的自磷酸化(大约1%)已经足够使得蛋白质本身完成交叉磷酸化并使蛋白达到一个稳定的磷酸化水平。第六章简单介绍了一些植内向整流钾离子通道KAT1的前期工作。更多还原

【Abstract】 Protein is one of the most important biomacromolecules for biological functions. Most of protein function is predicated on dynamics, and better understanding on significance of life can be achieved by mechanism studies of protein in physiological processes.. NMR spectroscopy is a power tool for studying protein dynamic and mechanism since a wide range of time scales. PYL10is one of the member of Arabidopsis thaliana soluble abscisic acid(ABA) receptor family (RCAR1/PYR/PYL) inhibiting its downstream signal factor, type2C protein phosphatase(PP2C). In this thesis, we employ some biological experimental methods together with NMR to investigate biological functionof PYL10. A conclusion that ABA plays an important role in the PP2C inhibiting function by PYLO, rather than "PYL10is an ABA-independent receptor" revealed by previous study, is generated according to our data. The dynamic assay of PYL10upon ABA binding was further investigated using liquid NMR relaxation and reduced spectral density function methods. The result shows ABA binding brings an increase of comformational entropy and protein flexibility of PYL10. Meanwhile, some other work which using mass spectrometry-based label-free quantitative method to perform site-specific quantitative analysis of Tyr574auto-phosphorylation of the Escherichia coli tyrosine kinase C-terminal catalytic domain(ETK-CTD) is also introduced in this thesis.In chapter one a brief introduction on the finding and classification of ABA receptor is given at the beginning. And then we describe the main content and function of the ABA signal pathway mediated by soluble RCAR1/PYR/PYL ABA receptor family. At the end of chapter one, we describe the structural changes in ABA perception and the mechanism of RCAR1/PYR/PYL family inhibiting phosphatase activity of PP2C.Chapter two is a brief review of methods on protein dynamic assay based on liquid NMR spin relaxation. Studies have shown that the combination of thermodynamics and dynamic can explain the protein-ligand or protein-protein integration mechanism reasonable.In chapter three, the chromogenic reaction of molybdate and phosphate was taken to investigate osmotic pressure influence on ABA receptor PYL10and PYL2inhibiting the PP2C(HAB1) phosphatase activity. The result shows PYL10inhibits HAB1phosphatase activity is ABA-dependent and would be influenced by BSA which is contrary to previously ABA-dependent conclusion. Further biochemical assay shows that PYL10and BSA wouldn’t form a stable complex.Based on the conclusion that BSA doesn’t influence the function of PYL10via direct interaction, a corollary that this effect may come from the dynamic changes of PYL10. To investigate this, in chapter four, a lot of experiments were taken including the backbone assignment, relaxation data collection and reduced spectral density function assay of free state PYL10(apo-PYL10) and ABA/PYL10complex and the titration assay, a conclusion that the increasing inhibition of PP2C activity by PYL10is the result of PYL10conformation entropy and flexibility increase during ABA binding was achieved.In chapter five, we used the label-free mass relative quantification assay and western-blot qualitative assay to investigate the phosphorylation level changes of ETK-CTD autophosphorylation site Tyr574and the total protein phosphorylation level respectively. The mass spectrum result shows that the phosphorylation level of ETK-CTD Tyr574reaches a low but stable rate(about1%) very quickly. However, the western-blot assay indicates the total protein phosphorylation level remains elevated eventhough Tyr574phosphorylation reaches the stable rate. These data strongly suggest that a low level of auto-phosphorylation is sufficient to trigger cross-phosphorylation to reach a steady state of total phosphorylation.The fourth chapter is a brief introduction of some preparatory work on plant inward rectifier potassium channels KAT1.更多还原