基于电子顺磁共振方法的膜蛋白结构解析以及功能研究

发布时间：2018-01-09 07:40

  本文关键词：基于电子顺磁共振方法的膜蛋白结构解析以及功能研究　出处：《中国科学技术大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 电子顺磁共振 膜蛋白 动力学 易趋性 距离测量 跨膜结构域 硫氰酸酶 干扰素诱导膜蛋白

【摘要】：通过位点特异性自旋标记电子顺磁共振方法能够获得生物大分子的动力学信息、易趋性信息和距离信息,近年来该方法已经被广泛的应用于生物大分子的结构功能研究中。在本论文中,我们使用电子顺磁共振方法对膜蛋白YgaP和IFITM3的结构功能进行研究,成功地解析了去污剂中全长YgaP膜蛋白的三维结构以及IFITM3蛋白的跨膜拓扑结构。论文由四个章节组成。第一章简要介绍了电子顺磁共振的基本理论、电子顺磁共振中的各种相互作用力以及电子顺磁共振谱仪的组成。第二章主要介绍电子顺磁共振方法在生物大分子中的应用。首先简要描述各种不同的自旋标记物和自旋标记方法。然后重点阐述位点特异性自旋标记电子顺磁共振技术的基本原理,并详细描述通过该方法得到蛋白质特定位点的动力学信息、易趋性信息以及位点之间的距离信息。最后还简要介绍了几个电子顺磁共振方法在膜蛋白以及核酸的结构功能研究中的实例。在第三章中我们综合使用连续波电子顺磁共振、脉冲电子顺磁共振以及刚体计算方法成功地解析了全长YgaP膜蛋白的三维结构。具体的来说,首先通过系统的连续波电子顺磁共振分析得到YgaP蛋白跨膜结构域的动力学数据和易趋性数据,进而获得YgaP蛋白跨膜结构域的二级结构特征。然后使用连续波电子顺磁共振和双电子-电子共振方法测量得到YgaP蛋白跨膜结构域中的距离信息,并用于刚体结构计算中,获得YgaP跨膜结构域的三维结构。而后,我们还运用双电子-电子共振方法测量得到YgaP跨膜结构域与水溶性硫氰酸酶结构域之间的长程距离约束,并结合硫氰酸酶结构域的液体核磁共振结构,通过刚体计算方法得到全长YgaP膜蛋白的三维结构。此外,我们还对YgaP膜蛋白结合硫氰酸酶反应产物SCN-前后的动力学、易趋性和距离变化进行检测,结果发现YgaP蛋白结合底物SCN-后会发生构象变化,揭示了大肠杆菌细胞膜上的YgaP蛋白转运硫氰酸根离子的机制。第四章中我们综合使用电子顺磁共振方法和核磁共振方法解析了IFITM3蛋白的跨膜结构域在去污剂DPC中的跨膜拓扑结构。连续波电子顺磁共振谱图分析和易趋性数据分析表明IFITM3蛋白的C末端存在一个长跨膜螺旋,N端疏水区域含有两个较短的膜内α螺旋,该结果证明IFITM3蛋白是一个Ⅱ型膜蛋白。我们还使用液体核磁共振方法对IFITM3蛋白的二级结构和主链弛豫进行分析,结果进一步证明IFITM3蛋白的Ⅱ型膜蛋白拓扑结构。该跨膜拓扑结构为IFITM3蛋白能够阻碍膜半融合的病毒抑制机理提供了结构基础。
[Abstract]:By site-specific spin labeling electron paramagnetic resonance method can obtain the kinetic information of biological macromolecules, easy taxis and distance information, in recent years, this method has been widely used in the structure and function of biological macromolecules in the study. In this thesis, we use electron paramagnetic resonance method was used to study the structure and function of membrane protein YgaP and IFITM3, successfully resolved three-dimensional structure of YgaP membrane protein and detergent in the full-length IFITM3 protein transmembrane topology. The thesis consists of four chapters. The first chapter briefly introduces the basic theory of electron paramagnetic resonance, electron spin magnetic resonance in interaction and electron paramagnetic resonance. The spectrometer. The second chapter mainly introduces the application of electron paramagnetic resonance method in biological macromolecules. Spin label and spin label method begins with a brief description of the various natural. After focusing on the basic principle of electron spin resonance technique for site-specific, and a detailed description of the dynamics of protein specific sites through this method, the distance information between information and easy taxis sites. Finally briefly introduces several examples of electron paramagnetic resonance method in membrane proteins as well as the structure and function of nucleic acid in the third chapter. We use continuous wave pulse electron paramagnetic resonance, electron paramagnetic resonance and rigid calculation method successfully resolved three-dimensional structure of full-length YgaP membrane protein. Specifically, first through the system of continuous wave electron paramagnetic resonance analysis of YgaP protein transmembrane domain and the kinetic data easy taxis data, two level structure characteristics so as to obtain a YgaP protein transmembrane domain. Then using continuous wave electron paramagnetic resonance and electron electron double resonance method The measured YgaP transmembrane domain in the distance information, and for the rigid structure calculation, the three-dimensional structure of YgaP transmembrane domain. Then, we use the double electron electron resonance method for measuring YgaP transmembrane domain and water soluble long-range distance constraint between rhodanese domains. Combined with the liquid NMR structure of rhodanese domain, 3-D structures of full-length YgaP membrane protein by rigid calculation method. In addition, we also combine the dynamics before and after the rhodanese reaction product SCN- of YgaP membrane protein, easy orientation and distance changes were detected, the results show that the conformational change of YgaP protein binding substrate SCN-, revealed the mechanism of Escherichia coli on the cell membrane YgaP protein transport thiocyanate ion. In the fourth chapter, we use electron paramagnetic analytical methods of magnetic resonance and nuclear magnetic resonance method IFITM3 The transmembrane domain protein in detergent DPC in transmembrane topology. Continuous wave electron paramagnetic resonance spectrum analysis and data analysis showed that the C terminal taxis IFITM3 protein has a long transmembrane helix, N terminal hydrophobic region containing two short film in alpha helix and the result shows that the IFITM3 protein is a type II membrane protein. We also use liquid NMR method on IFITM3 protein two level structure and main chain relaxation analysis, the results further prove that the type II membrane protein topology. IFITM3 proteins provide the structural basis for the transmembrane topology of IFITM3 protein can prevent membrane fusion of virus half inhibition mechanism.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q617
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本文编号：1400567