本文關鍵詞：人銅轉運蛋白1胞外蛋氨酸富集區(qū)與銀離子相互作用的研究　出處：《吉林大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 人銅轉運蛋白1 銀 相互作用 核磁共振波譜法 等溫滴定微量熱法

【摘要】：人類銅離子傳輸體蛋白1（hCtr1）是一種糖基化的膜蛋白，同時也是高度保守的銅離子傳輸體蛋白家族中的一員。它由190個氨基酸殘基組成，包含三個假定的跨膜區(qū)域、一個胞外的N端區(qū)域和一個胞內的C端區(qū)域。胞外N端區(qū)域有兩個蛋氨酸殘基富集的區(qū)域和兩個組氨酸殘基富集的區(qū)域，它們對于hCtr1蛋白傳輸銅離子和銀離子起重要作用。 在本論文中，我們運用圓二色譜法（CD）、核磁共振波譜法（NMR）以及等溫滴定微量熱法（ITC）等實驗方法研究了包含hCtr1蛋白N端蛋氨酸富集區(qū)M2在內的一個20肽及其突變體在SDS膠束溶液中與Ag(I)的配位方式以及配位過程中的熱力學性質。CD實驗表明原始肽WT在SDS膠束溶液中形成了部分螺旋結構。NMR實驗結果進一步表明肽在SDS膠束中形成了C端為螺旋、N端為靈活區(qū)域的結構。順磁性探針實驗表明，Gly1-Pro11區(qū)域內的氨基酸殘基位于膠束的表面，而Met12-Asn20氨基酸殘基則插入到膠束內部。在加入Ag(I)后，CD和NMR實驗結果都表明肽與Ag(I)發(fā)生了相互作用。肽段中所有Met的δ_(H_γ)和δ_(H_ε)均向低場方向移動，這說明Met與Ag(I)結合。結合2D-NOESY譜和2D-TOCSY譜我們發(fā)現(xiàn)第7位、第8位和第12位的Met提供了強的配位，第9位的Met提供了弱的配位，而第10位的Met不參與配位。 為進一步研究WT與Ag(I)的相互作用，我們進行了ITC實驗。通過ITC實驗我們得到了WT及各種突變體肽與Ag(I)在SDS膠束溶液中的相互作用的熱力學參數(shù)，如結合常數(shù)K、結合位點數(shù)n、結合反應焓變ΔH、熵變ΔS以及吉布斯自由能G。通過分析得到的熱力學參數(shù)，我們發(fā)現(xiàn)WT在SDS膠束中與Ag(I)以1:1的方式結合，結合常數(shù)為1.12×10~5M~(-1)，親和力比較弱，結合過程為焓驅動過程。與WT在SDS膠束溶液中與Ag(I)的配位結果相比，M7A、M8A和M12A的熱力學參數(shù)（主要是熵變和結合位點數(shù)）完全不同，M9A的熱力學參數(shù)則發(fā)生很大變化。ITC實驗結果進一步證明了NMR的結果，即殘基Met7、Met8、Met12（hCtr1中第40、41和45位蛋氨酸）在hCtr1-M2與Ag(I)的結合中起非常重要的作用，Met9（hCtr1中第42位蛋氨酸）的配位強度比它們弱些，，而Met10（hCtr1中第43位蛋氨酸）則不參與配位。 我們希望這些研究發(fā)現(xiàn)能為揭示銅轉運蛋白傳輸Ag(I)的機制提供一些有用的信息。
[Abstract]:Human copper ion transport protein 1hCtr1 is a glycosylated membrane protein and a member of the highly conserved copper ion transport protein family. It consists of 190 amino acid residues. There are three hypothetical transmembrane regions, one extracellular N-terminal region and one intracellular C-terminal region. There are two methionine residues enriched regions and two histidine residues enriched regions in the extracellular N-terminal region. They play an important role in the transport of copper and silver ions by hCtr1 protein. In this paper, we use circular dichroism (CD). Nuclear Magnetic Resonance Spectroscopy (NMR) and Isothermal Titration Microcalorimetry (ITC). A 20 peptide containing the N-terminal methionine rich region M2 of hCtr1 protein and its mutants were studied in SDS micelle solution. CD experiment showed that the original peptide WT formed partial helical structure in SDS micelle solution. NMR results further indicated that peptide formed in SDS micelle. It becomes a C-end spiral. The amino acid residues in Gly1-Pro11 region are located on the surface of micelles. The amino acid residues of Met12-Asn20 were inserted into the micelle. The results of CD and NMR experiments show that the peptide interacts with Agni, and all Met in the peptide region move in the direction of low field. Combined with 2D-NOESY and 2D-TOCSY spectra, we found that the Met at the 7th, 8th and 12th positions provide strong coordination. The 9 th bit Met provides weak coordination, while the 10 th position Met does not participate in the coordination. In order to further study the interaction between WT and Agni. We have carried out the ITC experiment and obtained the thermodynamic parameters of the interaction of WT and various mutant peptides with Agni in the SDS micelle solution, such as the binding constant K. The thermodynamic parameters are obtained by combining the number of sites n, the reaction enthalpy 螖 H, the entropy change 螖 S and the Gibbs free energy G. We found that WT binds with Agni in SDS micelles in the form of 1: 1 with a binding constant of 1.12 脳 10 ~ (5) M ~ (-1) and weak affinity. The binding process is a enthalpy driven process, which is compared with the coordination result of WT with Agni in SDS micelle solution. The thermodynamic parameters of M8A and M12A (mainly entropy change and binding site points) are completely different. The thermodynamic parameters of M9A vary greatly. The experimental results further prove the results of NMR. That is, the residues Met7, Met8, Met12hCtr1, and methionine at position 4041 and 45, play a very important role in the binding of hCtr1-M2 to Agni. The coordination intensity of the 42nd position methionine in Met9(hCtr1 is weaker than that in them, while the 43rd position methionine in Met10(hCtr1 is not involved in the coordination. We hope that these findings will provide some useful information for revealing the mechanism of copper transporter Ag-I.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R341

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