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研究生:蘇繼
研究生(外文):M.Sujith Kumar
論文名稱:探討人類蘋果酸酶二聚體與四聚體中間介面對結構穩定性之生物物理特性
論文名稱(外文):Biophysical characterization of dimer and tetramer interface interaction in stability of the human malic enzyme.
指導教授:洪慧芝洪慧芝引用關係
指導教授(外文):Hui-Chih Hung
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:98
語文別:英文
論文頁數:39
中文關鍵詞:蘋果酸&;#37238蘋果酸&;#37238
外文關鍵詞:malic enzymestability
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蘋果酸&;#37238;具有雙二聚體的四級結構,其中二聚體中間介面較四聚體中間介面更為緊密接觸。細胞質蘋果酸&;#37238; (c-NADP-ME) 異構型以穩定四聚體型式存在且不具有協同作用與異位調控的特性。在本篇研究中,我們分析人類細胞質蘋果酸&;#37238;之二聚體上-下與左-右型式的結構穩定性。挑選位於二聚體及四聚體中間介面之胺基酸進行於定點突變,分別為第51、90、139、142、568、572號胺基酸並獲得一系列人類細胞質蘋果酸&;#37238;之二聚體。我們構築二個單點突變 ( H142A, W572A ) , 三個雙點突變 (H51A/D90A, H51A/D139A, H142A/D568A ),表現於大腸桿菌並進行純化。藉由螢光與原二色光譜儀及 ANS 螢光分析技術,研究蛋白結構在熱變性與尿素誘發變性過程之去摺疊行為。從熱變性的結果得知,二聚體及四聚體中間介面之突變型的熱穩定性具有 &;#8710;Tm ~8-10 °C 的差異。推測由於二聚體中間介面之突變型具有較強的結合力。而在尿素誘發變性過程顯示二聚體中間介面之突變型的第一轉變階段位移至較低的尿素濃度。螢光實驗結果顯示破壞中間介面的結合力進而降低結構穩定性。從 ANS 螢光分析得知,二聚體中間介面之突變型的最大螢光強度出現於低尿素濃度。我們的研究證實人類細胞質蘋果酸&;#37238;之結構穩定性不受四聚體中間介面結合力破壞的影響。四聚體中間介面之突變型與野生型呈現相似的結構穩定性。然而,當破壞二聚體中間介面結合力則顯著喪失人類細胞質蘋果酸&;#37238;之結構穩定性且二聚體中間介面之突變型的結構穩定性低於野生型。以上結果顯示,相較於四聚體中間介面之結合力,二聚體中間介面之結合力在人類細胞質蘋果酸&;#37238;之結構穩定性中扮演著重要角色。

Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) isoform is non-cooperative and non-allosteric and exists as a stable tetramer. In this study, we analyzed the stability of up-and–down dimer and left-and-right dimers of human c-NADP-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME. Trp-572, His-142, Asp-568, His-51, Asp-90 and Asp-139 were selected as target sites for mutagenesis, because they are at the subunit interface. W572A, H142A, H142A/D568A, H51A/D90A and H51A/D139A mutant enzymes were constructed, expressed in Escherichia coli, and purified. We studied thermal and urea-induced unfolding using fluorescence and CD spectroscopy, as well as ANS fluorescence. Thus, the difference in thermo stability between tetramer and dimer interface mutants were ∆Tm ~8-10 °C. It is related to the strength of interaction between the dimer interface. Urea-induced unfolding also showed the first transistion shift towards lower urea concentration in dimer interface mutants. The Intrinsic fluorescence studies showed the decrease in stability due to the disruption of interface interaction. The ANS binding studies showed that the maximum intensity observed at lower concentration of urea in dimer interface mutants. Our studies revealed that the enzyme stability of c-NADP-ME is not affected by disruption of the tetramer interface. The analysis of tetramer interface mutants showed similar stability with wild type. However, disruption on the dimer interface showed significant loss of stability of c-NADP-ME and the analysis of the dimer interface mutants showed less stability than the wild type. These results indicate that the interaction between the dimer interface play an important role in the stability of c-NADP-ME than the interactions between the tetramer interface.

Contents…………………………………………………………………………………………………………….I
Table contents……………………………………………………………………………………………………III
Figure contents…………………………………………………………………………………………………...IV
Abbreviation……………………………………………………………………………………………………….V
Chinese abstract ………………………………………………………………………………………………….1
Abstract………………………………………………………………………………………………………........2
Introduction………………………………………………………………………………………………………...3
Materials and Methods……………………………………………………………………………………………7
Competent cell preparation………………………………………………………………………………….7
Plasmid DNA extraction…………………………………………………………………………..…………8
Transformation…………………………………………...………………………………………..…………8
Site directed mutagenesis…………………………………………………………………………………..9
Protein expression and purification……………………………………………….……………………….9
Thermal Induced unfolding by circular dichroism spectrometry………………………………...........10
Urea Induced unfolding by circular dichroism spectrometry…………………………………………...10
ANS titration………………..............................................................................................................11
Urea induced unfolding studies by Fluorescence Spectroscopy………………....…………………..12
ANS binding studies by Fluorescence Spectroscopy…………………………………………………..12
Results…………………………………………………………………………………………………….13
Expression and purification of the human c-NADP-ME WT and interface mutants…………13
Thermal denaturation of the human c-NADP-ME WT and interface mutants……………..…13
Urea denaturation of the human c-NADP-ME WT and interface mutants……………………14
Equilibrium Unfolding of human c-NADP-ME WT and Interface mutants by Urea………….14
Exposure of hydrophobic region of human c-NADP-ME WT and interface mutants during urea denaturation……………………………………………………………………………15
Discussion………………………………………………………………………………………………. 16
Reference………………………………………………………………………………………………..20
Tables……………………………………………………………………………………………………..25
Figures……………………………………………………………………………………………………30
Preparation of Reagents…………………………………………………………………………………38


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