發(fā)布時(shí)間：2018-03-05 07:49

  本文選題：魔角旋轉(zhuǎn)固體核磁共振　切入點(diǎn)：同位素標(biāo)記方法　出處：《中國(guó)科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：魔角旋轉(zhuǎn)固體核磁共振(Magic Angle Spinning Solid-state Nuclear Magnetic Resonance, MAS SSNMR)被認(rèn)為是一種適合用于研究難溶解、難結(jié)晶的蛋白質(zhì)結(jié)構(gòu)的有力手段。其中,膜蛋白是MAS SSNMR非常重要的研究對(duì)象之一。膜蛋白是生物體內(nèi)各種細(xì)胞活動(dòng)的主要參與者和執(zhí)行者,是一類極其重要的疏水性蛋白質(zhì)。目前市場(chǎng)上約70%的藥物靶點(diǎn)是膜蛋白。然而由于膜蛋白難以表達(dá)、疏水性及不易結(jié)晶等特點(diǎn),使得這一類蛋白質(zhì)的結(jié)構(gòu)研究極其困難。MAS SSNMR在研究膜蛋白結(jié)構(gòu)上有不可替代的四大優(yōu)勢(shì)：可以在天然膜環(huán)境或更接近天然膜環(huán)境的磷脂雙分子層上進(jìn)行膜蛋白結(jié)構(gòu)的研究、在理論上固體NMR信號(hào)的線寬不受蛋白分子量的影響、能夠提供膜蛋白動(dòng)力學(xué)及其與其它蛋白或者小分子的相互作用信息,因此,這一技術(shù)在膜蛋白結(jié)構(gòu)研究上具有很大的發(fā)展?jié)摿�。高分辨蛋白質(zhì)結(jié)構(gòu)的解析,尤其是對(duì)于大分子量的蛋白質(zhì)、多聚體的膜蛋白及具有多個(gè)結(jié)構(gòu)域的蛋白質(zhì)的結(jié)構(gòu)解析,依賴于大量的距離約束。然而,MAS SSNMR用于蛋白質(zhì)結(jié)構(gòu)研究的最大瓶頸正是很難獲得足夠數(shù)量的距離約束。其主要原因在于：(1)MAS SSNMR中譜峰重疊嚴(yán)重,很難準(zhǔn)確歸屬譜峰對(duì)應(yīng)的距離約束；(2)譜峰重疊難以通過(guò)增加實(shí)驗(yàn)維數(shù)來(lái)解決,如由二維實(shí)驗(yàn)到三維實(shí)驗(yàn),因?yàn)镸AS SSNMR常用的13C和15N檢測(cè)的靈敏度低,很難利用三維或四維實(shí)驗(yàn)測(cè)量距離；(3)更重要的是,由于自旋核本身的物理屬性限制,由自旋核之間偶極-偶極耦合作用較弱,獲得的長(zhǎng)程距離約束通常6 A。針對(duì)這些問(wèn)題,作者開(kāi)展了三方面的研究工作：實(shí)現(xiàn)了氨基酸選擇性反標(biāo)記和13C隔位標(biāo)記方法、發(fā)展了用于MAS SSNMR的順磁標(biāo)記-贗接觸位移技術(shù)(Pseudocontact shift, PCS)、結(jié)合多種同位素標(biāo)記方法和順磁標(biāo)記技術(shù)研究膜蛋白DAGK(diacylgycerol kinase)單體間的界面及三維結(jié)構(gòu)。首先,利用模型蛋白GB1,我們摸索了不同的氨基酸進(jìn)行反標(biāo)記的效率。使用葡萄糖作碳源,F、W、Y、I、L、K和T七種氨基酸可以同時(shí)被反標(biāo)記。使用[1,3-13C]-甘油或[2-13C]-甘油或[1-13C]-葡萄糖或[2-13C]-葡萄糖作碳源,我們?cè)谀さ鞍譊AGK上系統(tǒng)對(duì)比了不同碳源的隔位標(biāo)記效果差異,[1-13C]-葡萄糖和[2-13C]-葡萄糖的隔位標(biāo)記樣品分辨率略差于[1,3-13C]-甘油和[2-13C]-甘油隔位標(biāo)記的樣品分辨率,且信噪比遠(yuǎn)低于后者。對(duì)于膜蛋白樣品,使用[1,3-13C]-甘油和[2-13C]-甘油進(jìn)行隔位標(biāo)記是更好的選擇。然后,我們發(fā)展了用于MAS SSNMR的順磁標(biāo)記PCS方法來(lái)解析蛋白質(zhì)的三維結(jié)構(gòu)。在這項(xiàng)工作當(dāng)中,通過(guò)人為引入外源的磁各向異性順磁金屬離子,從MAS SSNMR譜圖中獲得PCS數(shù)據(jù),結(jié)合使用Rosetta的結(jié)構(gòu)計(jì)算方法,我們計(jì)算得到了一個(gè)高分辨率的蛋白質(zhì)三維結(jié)構(gòu)。PCS在提供距離約束上具有質(zhì)量高、效率高和數(shù)量多的優(yōu)勢(shì),很有潛力成為用于研究具有挑戰(zhàn)性地生物大分子的常規(guī)方法,比如研究膜蛋白及淀粉樣蛋白質(zhì)纖維的結(jié)構(gòu)。最后,我們結(jié)合多種同位素標(biāo)記方法及順磁標(biāo)記PRE (Paramagnetic relaxation enhancement)方法研究了膜蛋白DAGK在E.coli天然膜提取物中的四級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)。在這項(xiàng)工作中,我們找到了將穩(wěn)定的DAGK三聚體變性解聚成單體的方法,并能夠?qū)⒆冃缘腄AGK單體重新組裝成三聚體。通過(guò)相關(guān)實(shí)驗(yàn)驗(yàn)證,經(jīng)過(guò)變復(fù)性之后,DAGK的結(jié)構(gòu)未發(fā)生改變,且保留較好的酶活性。結(jié)合多種標(biāo)記方式和核磁方法,我們獲得了膜蛋白DAGK的四級(jí)結(jié)構(gòu)信息,證明DAGK在E.coli天然膜提取物中的四級(jí)結(jié)構(gòu)與其在去垢劑DPC和油脂立方相LCP中的有相同性也有明顯的差異性。在DAGK單體界面的研究過(guò)程中,我們使用了TEDOR、PDSD和分子間的PRE三種方法。通過(guò)對(duì)比,分子間的PRE是更適合用于研究膜蛋白單體間界面的方法,它具有核磁共振實(shí)驗(yàn)操作簡(jiǎn)單、分子間距離約束信號(hào)容易歸屬、長(zhǎng)程距離遠(yuǎn)和距離約束豐富的優(yōu)勢(shì),勢(shì)必會(huì)被更廣泛的應(yīng)用在膜蛋白的四級(jí)結(jié)構(gòu)研究中。
[Abstract]:MAS NMR (Magic Angle Spinning Solid-state Nuclear Magnetic Resonance, MAS SSNMR) is considered to be a suitable for studying the insoluble protein structure, powerful means difficult to crystallization. The membrane protein is very important to study the MAS SSNMR one of the objects. The membrane protein is the main participants and perform various activities of cell biology in person, is a kind of hydrophobic protein is extremely important. The current drug target market is about 70% of the membrane protein. However due to the hydrophobic membrane protein expression, and is not easy to crystallization, which makes the research structure of this class of proteins is extremely difficult four major advantages of.MAS SSNMR can not be replaced in the study membrane protein structure: can the study of membrane protein structure in natural membrane environment or closer to the natural environment of the membrane lipid bilayer, linewidth solid NMR signal in theory Not affected by the molecular weight of the protein, can interact with information, provide the kinetics of membrane proteins and other proteins or small molecules. Therefore, this technology has great potential in the study of membrane protein structure. High resolution protein structures, especially for high molecular weight protein, protein structure determination of membrane proteins multimers and multiple domains with the distance dependent constraints in large quantities. However, MAS SSNMR for the biggest bottleneck in protein structure research is difficult to obtain a sufficient number of distance constraint. The main reason lies in: (1) MAS SSNMR peaks overlap seriously, it is difficult to accurately attribution distance constraint peaks; (2) the overlap of the peaks to experiment by increasing the dimension to solve, such as from 2D to 3D experimental experiment, because the sensitivity of 13C and 15N detection used MAS SSNMR is low, it is difficult to use three or four The dimension measuring distance; (3) is more important, because the physical properties of nuclear spin is limited by nuclear spin between the dipole dipole coupling is weak, long distance constraints often get 6 A. to solve these problems, the author carried out the research work in three aspects: the selective anti amino acid markers and 13C compartment mark method for paramagnetic markers and pseudo contact technology MAS SSNMR development (Pseudocontact shift, PCS), combined with a variety of isotope labeling method and paramagnetic markers of membrane protein DAGK (diacylgycerol kinase) the interface between monomers and three-dimensional structure. Firstly, using the model protein GB1, we explored different amino acids efficiency of anti markers. Using glucose as carbon source, F, W, Y, I, L, K and T seven kinds of amino acids can also be anti mark. Use [1,3-13C]- or [2-13C]- or 1-13C]- glycerol glycerol or glucose [[2-13C]- Glucose as carbon source, we in the membrane protein DAGK system according to the comparison of different carbon sources by mark, [1-13C]- glucose and [2-13C]- glucose compartment labeled sample sample in [1,3-13C]- resolution slightly worse resolution of glycerol and [2-13C]- glycerol compartment markers, and the signal-to-noise ratio is much lower than the latter. The membrane protein samples, using [1,3-13C]- and [2-13C]- are the glycerol glycerol labeling is a better choice. Then, we developed a three-dimensional structure for paramagnetic labeling PCS method for the analysis of protein MAS SSNMR. In this work, the magnetic metal ions along the magnetic anisotropy by artificial introduction of exogenous PCS, data obtained from MAS SSNMR spectrum diagram the use of Rosetta, combined with the structure calculation method, we obtained a high resolution 3D protein structure of.PCS with high quality in distance constraint, high efficiency and quantity The advantages, it has a potential to be used in conventional methods of challenging large biological molecules, such as membrane protein and amyloid protein fiber structure. Finally, we combine a variety of isotope labeling method and magnetic marker PRE (Paramagnetic relaxation enhancement) method to study the four level structure in E.coli membrane in membrane of natural extracts protein DAGK and three level of structure. In this work, we will find a stable DAGK trimer modified method depolymerized into monomers, and DAGK monomers can be modified and re assembled into trimers. Through experimental verification, after denaturation and renaturation, the structure of DAGK is not changed, and reserved high enzymatic activity. Combined with a variety of markers and NMR method, we obtained four order structural information of membrane protein DAGK and prove the four level structure in E.coli membrane in DAGK and natural extracts in detergent Agent DPC and oil in cubic LCP with the same sex also have obvious differences. In the research process of DAGK single interface, we use TEDOR, PDSD and intermolecular PRE three methods. By contrast, the intermolecular PRE method is more suitable for the study of membrane protein monomer interface it has, the NMR experiment has the advantages of simple operation, the distance between the molecules constrained signal easy attribution, long distance and distance constraint rich advantages will be more widely applied in the research of four level structure of membrane proteins.

【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q617;O657.2

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本文編號(hào)：1569362