利用魔角旋转固体核磁共振解析蛋白质结构的标记方法和应用研究

发布时间：2018-03-05 07:49

本文选题：魔角旋转固体核磁共振　切入点：同位素标记方法　出处：《中国科学院研究生院(武汉物理与数学研究所)》2015年博士论文　论文类型：学位论文

【摘要】：魔角旋转固体核磁共振(Magic Angle Spinning Solid-state Nuclear Magnetic Resonance, MAS SSNMR)被认为是一种适合用于研究难溶解、难结晶的蛋白质结构的有力手段。其中,膜蛋白是MAS SSNMR非常重要的研究对象之一。膜蛋白是生物体内各种细胞活动的主要参与者和执行者,是一类极其重要的疏水性蛋白质。目前市场上约70%的药物靶点是膜蛋白。然而由于膜蛋白难以表达、疏水性及不易结晶等特点,使得这一类蛋白质的结构研究极其困难。MAS SSNMR在研究膜蛋白结构上有不可替代的四大优势：可以在天然膜环境或更接近天然膜环境的磷脂双分子层上进行膜蛋白结构的研究、在理论上固体NMR信号的线宽不受蛋白分子量的影响、能够提供膜蛋白动力学及其与其它蛋白或者小分子的相互作用信息,因此,这一技术在膜蛋白结构研究上具有很大的发展潜力。高分辨蛋白质结构的解析,尤其是对于大分子量的蛋白质、多聚体的膜蛋白及具有多个结构域的蛋白质的结构解析,依赖于大量的距离约束。然而,MAS SSNMR用于蛋白质结构研究的最大瓶颈正是很难获得足够数量的距离约束。其主要原因在于：(1)MAS SSNMR中谱峰重叠严重,很难准确归属谱峰对应的距离约束；(2)谱峰重叠难以通过增加实验维数来解决,如由二维实验到三维实验,因为MAS SSNMR常用的13C和15N检测的灵敏度低,很难利用三维或四维实验测量距离；(3)更重要的是,由于自旋核本身的物理属性限制,由自旋核之间偶极-偶极耦合作用较弱,获得的长程距离约束通常6 A。针对这些问题,作者开展了三方面的研究工作：实现了氨基酸选择性反标记和13C隔位标记方法、发展了用于MAS SSNMR的顺磁标记-赝接触位移技术(Pseudocontact shift, PCS)、结合多种同位素标记方法和顺磁标记技术研究膜蛋白DAGK(diacylgycerol kinase)单体间的界面及三维结构。首先,利用模型蛋白GB1,我们摸索了不同的氨基酸进行反标记的效率。使用葡萄糖作碳源,F、W、Y、I、L、K和T七种氨基酸可以同时被反标记。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,我们在膜蛋白DAGK上系统对比了不同碳源的隔位标记效果差异,[1-13C]-葡萄糖和[2-13C]-葡萄糖的隔位标记样品分辨率略差于[1,3-13C]-甘油和[2-13C]-甘油隔位标记的样品分辨率,且信噪比远低于后者。对于膜蛋白样品,使用[1,3-13C]-甘油和[2-13C]-甘油进行隔位标记是更好的选择。然后,我们发展了用于MAS SSNMR的顺磁标记PCS方法来解析蛋白质的三维结构。在这项工作当中,通过人为引入外源的磁各向异性顺磁金属离子,从MAS SSNMR谱图中获得PCS数据,结合使用Rosetta的结构计算方法,我们计算得到了一个高分辨率的蛋白质三维结构。PCS在提供距离约束上具有质量高、效率高和数量多的优势,很有潜力成为用于研究具有挑战性地生物大分子的常规方法,比如研究膜蛋白及淀粉样蛋白质纤维的结构。最后,我们结合多种同位素标记方法及顺磁标记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级结构和三级结构。在这项工作中,我们找到了将稳定的DAGK三聚体变性解聚成单体的方法,并能够将变性的DAGK单体重新组装成三聚体。通过相关实验验证,经过变复性之后,DAGK的结构未发生改变,且保留较好的酶活性。结合多种标记方式和核磁方法,我们获得了膜蛋白DAGK的四级结构信息,证明DAGK在E.coli天然膜提取物中的四级结构与其在去垢剂DPC和油脂立方相LCP中的有相同性也有明显的差异性。在DAGK单体界面的研究过程中,我们使用了TEDOR、PDSD和分子间的PRE三种方法。通过对比,分子间的PRE是更适合用于研究膜蛋白单体间界面的方法,它具有核磁共振实验操作简单、分子间距离约束信号容易归属、长程距离远和距离约束丰富的优势,势必会被更广泛的应用在膜蛋白的四级结构研究中。
[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.

【学位授予单位】：中国科学院研究生院(武汉物理与数学研究所)
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q617;O657.2

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