本文選題：核磁共振 + 蛋白質(zhì)二硫鍵異構(gòu)酶��； 參考：《中國科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所)》2016年碩士論文

【摘要】：蛋白質(zhì)二硫鍵異構(gòu)酶(PDI)是一種主要存在于內(nèi)質(zhì)網(wǎng)中的多功能蛋白,能夠氧化、還原和酶催化新生肽鏈的二硫鍵形成,幫助新生肽鏈折疊成正確的結(jié)構(gòu),以及抑制內(nèi)質(zhì)網(wǎng)中的無結(jié)構(gòu)蛋白聚集。PDI的晶體結(jié)構(gòu)已于近年獲得,但是PDI在溶液中的動(dòng)力學(xué)信息以及對(duì)PDI如何識(shí)別不同底物的分子機(jī)制仍不清楚。本論文第三章嘗試?yán)?9 F NMR的方法揭示PDI在溶液中結(jié)構(gòu)域運(yùn)動(dòng)以及PDI對(duì)不同底物的識(shí)別機(jī)制。結(jié)果顯示還原態(tài)PDI和氧化態(tài)PDI在溶液中的結(jié)構(gòu)柔性存在明顯差異。還原態(tài)PDI在溶液中存在兩種構(gòu)象交換,并且溫度的改變會(huì)引起兩種構(gòu)象比例的變化。氧化態(tài)PDI在溶液中的結(jié)構(gòu)則相對(duì)剛性。同時(shí)多肽底物和還原態(tài)PDI結(jié)合時(shí),會(huì)對(duì)構(gòu)象變化產(chǎn)生影響。但是不同底物結(jié)合對(duì)PDI構(gòu)象變化的影響不一樣,說明不同底物和PDI可能存在不同的結(jié)合模式。PDI在霍亂毒素致病過程中同樣發(fā)揮著重要作用,但是具體作用機(jī)制一直不明確,本文第四章通過NMR方法研究了PDI去折疊CTA的作用過程。我們發(fā)現(xiàn)只有還原態(tài)PDI能夠去折疊CTA,氧化態(tài)PDI和CTA之間雖然也存在弱相互作用,但是沒有去折疊CTA的功能；同時(shí)結(jié)構(gòu)上更類似于氧化態(tài)PDI打開狀態(tài)的突變體PDI C-S,即使在使用過量還原劑將CTA的二硫鍵還原的情況下,依然不能使CTA完全去折疊,說明半胱氨酸對(duì)PDI的結(jié)構(gòu)及其去折疊CTA的功能影響很大,緊湊且柔性大的結(jié)構(gòu)是PDI去折疊CTA的關(guān)鍵。PDI作為細(xì)胞內(nèi)質(zhì)網(wǎng)中重要的分子伴侶,與許多由無結(jié)構(gòu)蛋白聚集引起的疾病相關(guān)。天然無結(jié)構(gòu)蛋白α-synuclein在患者腦部的路易小體纖維化聚集,被認(rèn)為是引起帕金森癥的主要原因,體外實(shí)驗(yàn)證明,PDI能夠抑制α-synuclein的聚集,但其具體的分子機(jī)制還不清楚,研究PDI抑制α-synuclein聚集的具體機(jī)制對(duì)于帕金森癥的治療可能有所幫助。本論文第五章利用核磁共振方法研究了α-synuclein與PDI的相互作用,發(fā)現(xiàn)α-synuclein與PDI的主要結(jié)合位點(diǎn)位于α-synuclein的N端；然而將PDI所有的6個(gè)半胱氨酸突變成絲氨酸后,α-synuclein通過C末端與突變體PDI C-S結(jié)合；熒光實(shí)驗(yàn)結(jié)果表明突變體PDI C-S對(duì)α-synuclein纖維化聚集的抑制作用減弱,說明PDI抑制α-synuclein的纖維化聚集主要是通過與α-synuclein的N端殘基結(jié)合來實(shí)現(xiàn)的。本論文利用NMR在探測蛋白質(zhì)的動(dòng)力學(xué)信息和相互作用信息方面的優(yōu)勢,研究了PDI在溶液狀態(tài)下的構(gòu)象變化,以及PDI與帕金森癥、霍亂兩種病癥的關(guān)鍵蛋白的相互作用。
[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 偽 -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 偽 -synuclein can be inhibited, but its specific molecular mechanism is not clear. Studying the specific mechanism of PDI inhibiting 偽-synuclein aggregation may be helpful in the treatment of Parkinson's disease. In chapter 5, the interaction between 偽 -synuclein and PDI was studied by nuclear magnetic resonance. It was found that the main binding sites of 偽 -synuclein and PDI were at the N-terminal of 偽 -synuclein. However, after mutating all 6 cysteine from PDI to serine, 偽 -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 偽 -synuclein fibrosis aggregation is weakened. It is suggested that PDI inhibits the accumulation of 偽 -synuclein mainly by binding to the N-terminal residues of 偽 -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.
【學(xué)位授予單位】：中國科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O629.73

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本文編號(hào)：1812909