人血清白蛋白与配体之间相互作用的核磁共振研究

发布时间：2018-03-23 06:20

本文选题：结合位点　切入点：人血清白蛋白　出处：《中国科学院研究生院(武汉物理与数学研究所)》2016年硕士论文　论文类型：学位论文

【摘要】：人血清白蛋白(Human serum albumin)是人体内循环系统中含量最高的蛋白质,约占血清中蛋白含量的60%,通常健康成年人的血清中白蛋白的浓度约为50mg/mL(-600μM)。血清白蛋白的主要作用是结合并传递各种药物以及内源性代谢物至人体的各个器官组织。白蛋白体积较大,结构复杂,含有585个氨基酸残基,相对质量为66 kDa,其单体由三个结构域组成,其中每个结构域又含有两个子结构域。因此,在白蛋白的表面会形成多个结合位点,如脂肪酸结合位点,金属离子结合位点等。其中有两个主要的药物结合位点,称之为Sudlow's site Ⅰ和Sudlow's site Ⅱ。而药物在体内的药效药性,以及在血液中的有效浓度等,都与白蛋白上的这两个结合位点密切相关。白蛋白上还有四个主要的金属离子结合位点,其中Site A为锌离子的首要结合位点。而血清中的锌离子大部分都结合在白蛋白上,因此对锌离子结合位点的研究具有很高的重要性。晶体衍射是目前蛋白质研究中主要的研究方法。然而,结晶过程以及样品的配制需要消耗大量的时间,且繁琐复杂。核磁共振技术作为一种新兴的无损技术在蛋白质的分析领域得到了广泛的应用。核磁共振技术最大的优势在于方便快捷,我们无需再对样品进行分离预处理,.也无需破坏样品的完整性,从而大大降低了分离样品所带来的耗时和误差。由于白蛋白体积过于庞大,氨基酸之间互相重叠包裹,因此普通的一维氢谱不仅谱峰变宽严重,而且谱峰之间相互重叠,大大的影响了我们对谱图的分析。而常规的横向弛豫滤波技术虽然可以滤掉宽包信号,但同时也抑制掉了一些重要的信息。本课题中,我们通过引进新的谱编辑技术,可直接观测天然丰度白蛋白的相互作用信息。(1) 我们首先建立了基于辐射阻尼效应的WaterLOGSY谱编辑技术(RD-WaterLOGSY),通过该技术增强白蛋白信号,然后通过引入横向弛豫加权(T2W),滤掉白蛋白谱峰上的一些弛豫时间较短的宽包信号,保留白蛋白表面与水分子具有快速交换或者强NOE的信号。结合常规二维TOCSY谱,我们发现该方法所观察到的血清白蛋白信号主要来自于白蛋白表面的组氨酸。(2) 基于以上所建立的T2W-RD-WaterLOGSY技术,我们可观察离子与白蛋白的相互作用。我们发现在改变溶液的pH值时,T2W-RD-WaterLOGSY技术所观测的谱图上低场区某些信号化学位移会发生明显的移动,据此我们发现可以利用化学位移的相对变化来判断溶液pH的变化。另外,当向溶液中逐步加入金属锌离子时,T2W-RD-WaterLOGSY谱图上某些峰强度的变化与锌离子浓度的变化成线性关系,表明T2W-RD-WaterLOGSY技术可以对锌离子的结合起到指示的作用。(3) 基于以上T2W-RD-WaterLOGSY技术,我们可以还可以快速观察药物与白蛋白的相互作用,判断作用位点信息。我们向白蛋白溶液中加入了两种不同的药物,观察随着药物浓度的增加,白蛋白信号的变化情况。发现该技术所观测到的低场区的某些信号的化学位移和强度与特定药物结合位点有关：当药物结合在Sudlow's site Ⅰ时,这些低场区信号的强度发生改变,而当药物结合在Sudlow's site Ⅱ时,这些信号并没有发生明显变化。(4) 我们利用T2W-RD-WaterLOGSY技术对血清中白蛋白进行了检测,发现该技术同样可以选择性检测血清中的白蛋白,消除其他小分子信号的影响。
[Abstract]:Human serum albumin (Human serum albumin) is a protein content in human body in the circulatory system of the highest, accounting for about 60% of the protein content in serum, serum albumin concentration generally healthy adults in approximately 50mg/mL (-600 M). The main function of serum albumin is a combination of various drugs and endogenous metabolites and transfer to human bodies organ albumin. Large volume, complex structure, containing 585 amino acid residues, the relative quality of 66 kDa, the monomer consisting of three domains, each domain contains two sub domains. Because of this, the formation of multiple binding sites on albumin surface, such as fatty acid binding sites. The metal ion binding sites. There are two main drug binding site, called the Sudlow's site I and Sudlow's II and site. In vivo efficacy of drug resistance, and the effective concentration in the blood Etc., are closely related with these two albumin binding sites. There are four main metal ion binding sites on albumin, which Site A is the primary binding sites of zinc ions and zinc ions in serum. Most with albumin, so it has a high importance of a point of zinc ion. Crystal diffraction is the main research method of the protein research. However, the crystallization process and the sample preparation need to consume a large amount of time, cumbersome and complex. The nuclear magnetic resonance technology as a new nondestructive technique has been widely applied in the field of protein analysis. The advantage of NMR technology is the most convenient. We need no separation of the samples pretreatment. There is no need to destroy the integrity of the sample, thereby greatly reducing the sample separation caused by the time-consuming and error. Due to the volume of albumin The amino acid is too large, overlapping parcels, so the ordinary one-dimensional hydrogen peak broadening spectrum not only seriously, and spectral overlapping peaks, greatly affected our analysis of the spectrum. Although conventional T2 filtering can filter out Henan wide packet signal, but also suppress some important the information. In this paper, we introduce a new spectral editing technology, interaction information direct observation of natural abundance albumin. (1) we first established the radiation damping effect of WaterLOGSY spectral editing technology based on (RD-WaterLOGSY), through the enhancement of albumin signal, and then through the introduction of transverse relaxation weighted (T2W), to filter out some of the albumin peak relaxation time shorter width packet signal, signal reservation albumin surface and water molecule has rapid exchange or strong NOE. Combined with the conventional 2D TOCSY spectrum, we found that Serum albumin is the signal observed mainly from albumin surface histidine. (2) set up above based on T2W-RD-WaterLOGSY technology, we can observe the interaction between ions and albumin. We found that the change in the pH value of the solution, T2W-RD-WaterLOGSY technology, the observed spectra on the low field region some chemical shift signal will obviously move, we found that the relative change can be used to determine the chemical shift variation of the solution of pH. In addition, when the solution to gradually add zinc ions, changes of T2W-RD-WaterLOGSY spectrum changes some peak intensity on the map and the linear relationship between the concentration of zinc ions, showed that combination of T2W-RD-WaterLOGSY technology for zinc ion pointing to the role. (3) the above interaction based on T2W-RD-WaterLOGSY technology, we can also observe fast drug and albumin, sentenced Off site information. We add two different drugs to albumin solution, observed with the increase of drug concentration, the changes of albumin signal. This technology observed in the low field signal region of some of the chemical shift and intensity and specific drug binding sites for drug binding in Sudlow's site: when I was the low field region, the intensity of the signal changes, and when the drug combination in Sudlow's site II, these signals were not changed obviously. (4) we use T2W-RD-WaterLOGSY technology to detect the serum albumin, this technology can also be selectively detected in serum albumin, the elimination of other small molecule signal.

【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】：硕士
【学位授予年份】：2016
【分类号】：O657.2

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