本文選題：CRAC通道 + Orai��； 參考：《四川師范大學(xué)》2017年碩士論文

【摘要】：Ca~(2+)是真核細(xì)胞重要的細(xì)胞內(nèi)信使。細(xì)胞內(nèi)Ca~(2+)濃度的變化是許多生理進(jìn)程所必需的。鈣庫(kù)操縱的鈣通道(SOCs)是鈣離子流入細(xì)胞內(nèi)的主要的方式,其中最著名最具特色的是鈣離子釋放激活鈣離子(CRAC)通道。CRAC通道調(diào)節(jié)許多基本的細(xì)胞功能,包括基因表達(dá)、細(xì)胞增殖、細(xì)胞分泌等。在近十幾年里,作為CRAC通道的Orai蛋白和作為內(nèi)質(zhì)網(wǎng)鈣離子感受器的STIM蛋白的發(fā)現(xiàn)是理解CRAC通道的機(jī)制和功能取得極大進(jìn)步的基礎(chǔ)。當(dāng)內(nèi)質(zhì)網(wǎng)鈣庫(kù)釋放后,STIM蛋白發(fā)生一系列的構(gòu)象變化與Orai蛋白相互作用,從而開放CRAC通道,允許細(xì)胞外的鈣離子流入細(xì)胞內(nèi)。但CRAC通道門控的分子機(jī)制仍然知之甚少。為了進(jìn)一步揭示CRAC通道門控的分子機(jī)制,我們表達(dá)并分離純化了果蠅源的Orai蛋白和STIM蛋白不同片段。在過程中對(duì)STIM蛋白不同片段的分離純化條件進(jìn)行了優(yōu)化,對(duì)STIM蛋白不同片段進(jìn)行了功能測(cè)試,并最終獲得了較穩(wěn)定并具有功能的STIM蛋白片段,同時(shí)對(duì)純化的Orai蛋白和STIM蛋白片段進(jìn)行了等溫滴定測(cè)定其相互作用。我們發(fā)現(xiàn)Orai蛋白與STIM蛋白片段親和力較強(qiáng),這為后續(xù)通過單分子熒光能量轉(zhuǎn)移技術(shù)和蛋白技術(shù)共結(jié)晶進(jìn)一步的研究?jī)傻鞍椎南嗷プ饔玫臉?gòu)象變化奠定了基礎(chǔ)。
[Abstract]:Ca~(2) is an important intracellular messenger in eukaryotic cells. Changes in intracellular Ca~(2 concentration are necessary for many physiological processes. Calcium channel SOCsis the main way of calcium ions flowing into cells. The most famous and characteristic is calcium ion release activated calcium ion CRAC.CRAC channel regulates many basic cellular functions, including gene expression and cell proliferation. Cell secretion, etc. In recent years, the discovery of Orai protein as CRAC channel and STIM protein as endoplasmic reticulum calcium receptor has been the basis of great progress in understanding the mechanism and function of CRAC channel. When the endoplasmic reticulum calcium reservoir was released, a series of conformational changes occurred between STIM protein and Orai protein, thus opening up the CRAC channel and allowing extracellular calcium ions to flow into the cell. However, little is known about the molecular mechanism of CRAC channel gating. In order to further reveal the molecular mechanism of CRAC channel gating, we expressed and purified different fragments of Orai and STIM proteins from Drosophila melanogaster. In the process, the conditions of isolation and purification of different STIM protein fragments were optimized, and the functional tests of different STIM protein fragments were carried out. Finally, stable and functional STIM protein fragments were obtained. At the same time, the interaction between purified Orai protein and STIM protein was determined by isothermal titration. We found that Orai protein has strong affinity with STIM protein fragment, which lays a foundation for further study on conformation change of interaction between two proteins by single molecule fluorescence energy transfer technique and protein technology.
【學(xué)位授予單位】：四川師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q51

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