本文關(guān)鍵詞：基于電子順磁共振方法的膜蛋白結(jié)構(gòu)解析以及功能研究　出處：《中國科學(xué)技術(shù)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 電子順磁共振 膜蛋白 動(dòng)力學(xué) 易趨性 距離測量 跨膜結(jié)構(gòu)域 硫氰酸酶 干擾素誘導(dǎo)膜蛋白

【摘要】：通過位點(diǎn)特異性自旋標(biāo)記電子順磁共振方法能夠獲得生物大分子的動(dòng)力學(xué)信息、易趨性信息和距離信息,近年來該方法已經(jīng)被廣泛的應(yīng)用于生物大分子的結(jié)構(gòu)功能研究中。在本論文中,我們使用電子順磁共振方法對(duì)膜蛋白YgaP和IFITM3的結(jié)構(gòu)功能進(jìn)行研究,成功地解析了去污劑中全長YgaP膜蛋白的三維結(jié)構(gòu)以及IFITM3蛋白的跨膜拓?fù)浣Y(jié)構(gòu)。論文由四個(gè)章節(jié)組成。第一章簡要介紹了電子順磁共振的基本理論、電子順磁共振中的各種相互作用力以及電子順磁共振譜儀的組成。第二章主要介紹電子順磁共振方法在生物大分子中的應(yīng)用。首先簡要描述各種不同的自旋標(biāo)記物和自旋標(biāo)記方法。然后重點(diǎn)闡述位點(diǎn)特異性自旋標(biāo)記電子順磁共振技術(shù)的基本原理,并詳細(xì)描述通過該方法得到蛋白質(zhì)特定位點(diǎn)的動(dòng)力學(xué)信息、易趨性信息以及位點(diǎn)之間的距離信息。最后還簡要介紹了幾個(gè)電子順磁共振方法在膜蛋白以及核酸的結(jié)構(gòu)功能研究中的實(shí)例。在第三章中我們綜合使用連續(xù)波電子順磁共振、脈沖電子順磁共振以及剛體計(jì)算方法成功地解析了全長YgaP膜蛋白的三維結(jié)構(gòu)。具體的來說,首先通過系統(tǒng)的連續(xù)波電子順磁共振分析得到Y(jié)gaP蛋白跨膜結(jié)構(gòu)域的動(dòng)力學(xué)數(shù)據(jù)和易趨性數(shù)據(jù),進(jìn)而獲得YgaP蛋白跨膜結(jié)構(gòu)域的二級(jí)結(jié)構(gòu)特征。然后使用連續(xù)波電子順磁共振和雙電子-電子共振方法測量得到Y(jié)gaP蛋白跨膜結(jié)構(gòu)域中的距離信息,并用于剛體結(jié)構(gòu)計(jì)算中,獲得YgaP跨膜結(jié)構(gòu)域的三維結(jié)構(gòu)。而后,我們還運(yùn)用雙電子-電子共振方法測量得到Y(jié)gaP跨膜結(jié)構(gòu)域與水溶性硫氰酸酶結(jié)構(gòu)域之間的長程距離約束,并結(jié)合硫氰酸酶結(jié)構(gòu)域的液體核磁共振結(jié)構(gòu),通過剛體計(jì)算方法得到全長YgaP膜蛋白的三維結(jié)構(gòu)。此外,我們還對(duì)YgaP膜蛋白結(jié)合硫氰酸酶反應(yīng)產(chǎn)物SCN-前后的動(dòng)力學(xué)、易趨性和距離變化進(jìn)行檢測,結(jié)果發(fā)現(xiàn)YgaP蛋白結(jié)合底物SCN-后會(huì)發(fā)生構(gòu)象變化,揭示了大腸桿菌細(xì)胞膜上的YgaP蛋白轉(zhuǎn)運(yùn)硫氰酸根離子的機(jī)制。第四章中我們綜合使用電子順磁共振方法和核磁共振方法解析了IFITM3蛋白的跨膜結(jié)構(gòu)域在去污劑DPC中的跨膜拓?fù)浣Y(jié)構(gòu)。連續(xù)波電子順磁共振譜圖分析和易趨性數(shù)據(jù)分析表明IFITM3蛋白的C末端存在一個(gè)長跨膜螺旋,N端疏水區(qū)域含有兩個(gè)較短的膜內(nèi)α螺旋,該結(jié)果證明IFITM3蛋白是一個(gè)Ⅱ型膜蛋白。我們還使用液體核磁共振方法對(duì)IFITM3蛋白的二級(jí)結(jié)構(gòu)和主鏈弛豫進(jìn)行分析,結(jié)果進(jìn)一步證明IFITM3蛋白的Ⅱ型膜蛋白拓?fù)浣Y(jié)構(gòu)。該跨膜拓?fù)浣Y(jié)構(gòu)為IFITM3蛋白能夠阻礙膜半融合的病毒抑制機(jī)理提供了結(jié)構(gòu)基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q617
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本文編號(hào)：1400567