兰州大学机构库 >生命科学学院
电镜单颗粒技术解析蛋白质结构
Alternative Titlestructural studies of proteins by single particle EM
欧阳燕
Thesis Advisor吉尚戎
2016-06-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword电镜 单颗粒 三维重构 KlUA AMPK
Abstract近年来,电镜单颗粒技术迅速发展,成为结构生物学研究的一项主要技术手段。本论文介绍电镜单颗粒技术在两种蛋白质结构研究中的应用。在论文第一部分中,我们通过电镜单颗粒技术解析了尿素氨解酶的结构。尿素是很多含氮生物分子 (如精氨酸、嘌呤或酰脲等)的代谢产物,可继续被尿素氨解酶( urea Amidolyase, UA)转化为无机氨而重新纳入合成代谢,以实现氮元素在生物圈中的循环利用。 尿素氨解酶由尿素羧化酶(UC)结构域和脲基甲酸水解酶(AH)结构域组成,分两步反应实现尿素分解。 UA 是一个多结构域具有种酶活的蛋白,在发挥功能过程中需要各结构域彼此间的密切协同以在结构域间完成中间产物/底物的传递。目前 UC与AH结构域各自的晶体已被解析,但前者仅捕捉到 BCCP与CT结合的构象,而UA蛋白的整体构架及工作过程中介导结域间通讯的构象变化还不清楚。 本研究通过电镜单颗粒三维重构技术,解析UA全酶的分子构架及寡聚机制,为进一步研究UA发挥酶活过程中主要功能态 酶活过程中主要功能态的构象 变化机制打下基础。 在论文的第二部分,我们对通过电镜单颗粒技术解析的AMPK蛋白结构与晶体结构之间的架差异进行比较分析。结果表明,不同的制样条件下,通过电镜重构得到的AMPK结构模型的构架并无明显差异。进一步的分析表明,KD和CBM的分子内运动可能是由于分子内灵活的柔性连接序列以及可塑的分子间排列方式导致,显示出AMPK蛋白分子结构的可塑性与复杂性。
Other AbstractEM single particle technique has been developed vastly to become a mainstream structural biology method in recent years. In this dissertation two proteins were investigated via EM single particle analysis. In part І, the structure of Urea Amidolyase holoenzyme was solved. Urea is a metabolite of many nitrogen-containing biological molecules biological molecules, which may be converted to inorganic ammonia by Urea (UA) to reenter the anabolic pathway. By this way nitrogen is recycled in the bio-sphere. UA is composed of urea carboxylase ( UC) and allophanate hydrolase(AH) breakinging down urea in two steps.Crystal structures of UC and AH have been resolved now, with the former in a conformation of BCCP-biotin binding to CT active site. However, the architecture of UA holoenzyme is still unknown and the conformational changes in the process of activity unravelled. In this study,we determine the architecture and oligomeric mechanism of UA holoenzyme by EM single particle for further investigation the conformational changes of UA in the activation process . In part II, the architectural plasticity of AMPK protein revealed by single particle EM and X-ray crystallography was analyzed.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221539
Collection生命科学学院
Recommended Citation
GB/T 7714
欧阳燕. 电镜单颗粒技术解析蛋白质结构[D]. 兰州. 兰州大学,2016.
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