人铜转运蛋白1胞外蛋氨酸富集区与银离子相互作用的研究

发布时间：2018-01-16 16:16

本文关键词：人铜转运蛋白1胞外蛋氨酸富集区与银离子相互作用的研究　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：人类铜离子传输体蛋白1（hCtr1）是一种糖基化的膜蛋白，同时也是高度保守的铜离子传输体蛋白家族中的一员。它由190个氨基酸残基组成，包含三个假定的跨膜区域、一个胞外的N端区域和一个胞内的C端区域。胞外N端区域有两个蛋氨酸残基富集的区域和两个组氨酸残基富集的区域，它们对于hCtr1蛋白传输铜离子和银离子起重要作用。在本论文中，我们运用圆二色谱法（CD）、核磁共振波谱法（NMR）以及等温滴定微量热法（ITC）等实验方法研究了包含hCtr1蛋白N端蛋氨酸富集区M2在内的一个20肽及其突变体在SDS胶束溶液中与Ag(I)的配位方式以及配位过程中的热力学性质。CD实验表明原始肽WT在SDS胶束溶液中形成了部分螺旋结构。NMR实验结果进一步表明肽在SDS胶束中形成了C端为螺旋、N端为灵活区域的结构。顺磁性探针实验表明，Gly1-Pro11区域内的氨基酸残基位于胶束的表面，而Met12-Asn20氨基酸残基则插入到胶束内部。在加入Ag(I)后，CD和NMR实验结果都表明肽与Ag(I)发生了相互作用。肽段中所有Met的δ_(H_γ)和δ_(H_ε)均向低场方向移动，这说明Met与Ag(I)结合。结合2D-NOESY谱和2D-TOCSY谱我们发现第7位、第8位和第12位的Met提供了强的配位，第9位的Met提供了弱的配位，而第10位的Met不参与配位。为进一步研究WT与Ag(I)的相互作用，我们进行了ITC实验。通过ITC实验我们得到了WT及各种突变体肽与Ag(I)在SDS胶束溶液中的相互作用的热力学参数，如结合常数K、结合位点数n、结合反应焓变ΔH、熵变ΔS以及吉布斯自由能G。通过分析得到的热力学参数，我们发现WT在SDS胶束中与Ag(I)以1:1的方式结合，结合常数为1.12×10~5M~(-1)，亲和力比较弱，结合过程为焓驱动过程。与WT在SDS胶束溶液中与Ag(I)的配位结果相比，M7A、M8A和M12A的热力学参数（主要是熵变和结合位点数）完全不同，M9A的热力学参数则发生很大变化。ITC实验结果进一步证明了NMR的结果，即残基Met7、Met8、Met12（hCtr1中第40、41和45位蛋氨酸）在hCtr1-M2与Ag(I)的结合中起非常重要的作用，Met9（hCtr1中第42位蛋氨酸）的配位强度比它们弱些，，而Met10（hCtr1中第43位蛋氨酸）则不参与配位。我们希望这些研究发现能为揭示铜转运蛋白传输Ag(I)的机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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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