PDI构象变化及与其它蛋白质相互作用的NMR研究

发布时间：2018-04-28 00:01

本文选题：核磁共振 + 蛋白质二硫键异构酶　；参考：《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

【摘要】：蛋白质二硫键异构酶(PDI)是一种主要存在于内质网中的多功能蛋白,能够氧化、还原和酶催化新生肽链的二硫键形成,帮助新生肽链折叠成正确的结构,以及抑制内质网中的无结构蛋白聚集。PDI的晶体结构已于近年获得,但是PDI在溶液中的动力学信息以及对PDI如何识别不同底物的分子机制仍不清楚。本论文第三章尝试利用19 F NMR的方法揭示PDI在溶液中结构域运动以及PDI对不同底物的识别机制。结果显示还原态PDI和氧化态PDI在溶液中的结构柔性存在明显差异。还原态PDI在溶液中存在两种构象交换,并且温度的改变会引起两种构象比例的变化。氧化态PDI在溶液中的结构则相对刚性。同时多肽底物和还原态PDI结合时,会对构象变化产生影响。但是不同底物结合对PDI构象变化的影响不一样,说明不同底物和PDI可能存在不同的结合模式。PDI在霍乱毒素致病过程中同样发挥着重要作用,但是具体作用机制一直不明确,本文第四章通过NMR方法研究了PDI去折叠CTA的作用过程。我们发现只有还原态PDI能够去折叠CTA,氧化态PDI和CTA之间虽然也存在弱相互作用,但是没有去折叠CTA的功能；同时结构上更类似于氧化态PDI打开状态的突变体PDI C-S,即使在使用过量还原剂将CTA的二硫键还原的情况下,依然不能使CTA完全去折叠,说明半胱氨酸对PDI的结构及其去折叠CTA的功能影响很大,紧凑且柔性大的结构是PDI去折叠CTA的关键。PDI作为细胞内质网中重要的分子伴侣,与许多由无结构蛋白聚集引起的疾病相关。天然无结构蛋白α-synuclein在患者脑部的路易小体纤维化聚集,被认为是引起帕金森症的主要原因,体外实验证明,PDI能够抑制α-synuclein的聚集,但其具体的分子机制还不清楚,研究PDI抑制α-synuclein聚集的具体机制对于帕金森症的治疗可能有所帮助。本论文第五章利用核磁共振方法研究了α-synuclein与PDI的相互作用,发现α-synuclein与PDI的主要结合位点位于α-synuclein的N端；然而将PDI所有的6个半胱氨酸突变成丝氨酸后,α-synuclein通过C末端与突变体PDI C-S结合；荧光实验结果表明突变体PDI C-S对α-synuclein纤维化聚集的抑制作用减弱,说明PDI抑制α-synuclein的纤维化聚集主要是通过与α-synuclein的N端残基结合来实现的。本论文利用NMR在探测蛋白质的动力学信息和相互作用信息方面的优势,研究了PDI在溶液状态下的构象变化,以及PDI与帕金森症、霍乱两种病症的关键蛋白的相互作用。
[Abstract]:Protein disulfide isomerase (PDI) is a multifunctional protein that mainly exists in the endoplasmic reticulum. It can oxidize, reduce and catalyze the formation of disulfide bonds of the new peptide chain, helping the new peptide chain to fold into the correct structure. However, the kinetic information of PDI in solution and the molecular mechanism of how PDI can recognize different substrates have been obtained in recent years. In the third chapter, we try to reveal the movement of PDI domain in solution and the recognition mechanism of different substrates by PDI using 19F NMR method. The results show that there are obvious differences in structural flexibility between reduced PDI and oxidized PDI in solution. There are two conformation exchanges in the solution of reduced PDI, and the change of temperature will cause the change of the ratio of the two conformations. The structure of oxidized PDI in solution is relatively rigid. At the same time, when the peptide substrate binds to the reduced PDI, it will affect the conformation change. However, the effects of substrate binding on the conformation change of PDI are different, indicating that different substrate and PDI may have different binding modes. PDI also plays an important role in the pathogenesis of cholera toxin, but the specific mechanism is not clear. In chapter 4, the process of PDI unfolding CTA is studied by NMR method. It is found that only the reduced PDI can unfold the CTA, and that there is a weak interaction between the oxidized PDI and the CTA, but it does not have the function of unfolding the CTA. At the same time, the structure is more similar to that of PDI C-S, which is more similar to the open state of oxidized PDI. Even when the disulfide bond of CTA is reduced by excessive reductant, the CTA can not be completely unfolded. It is suggested that cysteine has a great influence on the structure of PDI and its function of unfolding CTA. The compact and flexible structure is the key of PDI unfolding CTA. PDI is an important molecular chaperone in the endoplasmic reticulum of cells. It is associated with many diseases caused by agglomeration of unstructured proteins. The accumulation of 伪 -synuclein in the brain of patients with Parkinson's disease is thought to be the main cause of Parkinson's disease. In vitro experiments have shown that 伪 -synuclein can be inhibited, but its specific molecular mechanism is not clear. Studying the specific mechanism of PDI inhibiting 伪-synuclein aggregation may be helpful in the treatment of Parkinson's disease. In chapter 5, the interaction between 伪 -synuclein and PDI was studied by nuclear magnetic resonance. It was found that the main binding sites of 伪 -synuclein and PDI were at the N-terminal of 伪 -synuclein. However, after mutating all 6 cysteine from PDI to serine, 伪 -synuclein binds to the mutant PDI C-S via C-terminal, and the fluorescence results show that the inhibitory effect of PDI C-S on 伪 -synuclein fibrosis aggregation is weakened. It is suggested that PDI inhibits the accumulation of 伪 -synuclein mainly by binding to the N-terminal residues of 伪 -synuclein. Based on the advantage of NMR in detecting protein kinetic information and interaction information, the conformation changes of PDI in solution state and the interaction between PDI and key proteins of Parkinson's disease and cholera were studied.
【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】：O629.73

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