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研究生:胡書銘
研究生(外文):Su-ming Hu
論文名稱:七萬道爾吞熱休克同源蛋白(Hsc70)碳端30-kDa區間之分析
論文名稱(外文):Characterization of the C-terminal 30-kDa Domain of 70-kDa Heat Shock Cognate Protein (Hsc70)
指導教授:王群王群引用關係
指導教授(外文):Chung Wang
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:85
中文關鍵詞:蛋白監護重新折疊受質結合
外文關鍵詞:chaperonerefoldingsubstrate bingding
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七十萬道爾吞熱休克同源蛋白(hsc70;70-kDa heat shock cognate protein)執行分子監護(molecular chaperone)功能需和未折疊蛋白結合,然後釋出。結合及釋出的循環反應受ADP/ATP調控。雖然hsc70氮端44-kDa的ATP水解酶區間之後的18-kDa可以結合短胜肽受質,但18-kDa無法和多胜肽受質形成穩定的複合體。我們利用大腸桿菌表現大白鼠hsc70的各種片段,發現包含了18-kDa和碳端10-kDa的30-kDa可以穩定結合模型多胜肽受質 S-carboxymethyl-alpha-lactalbumin (CMLA),也就是說30-kDa區間可穩定結合多胜肽受質必需有10-kDa片段。
分析18-kDa和10-kDa介面突變的突變株hsc70(L399P) 和hsc70(R447G)的結果顯示,它們的二級結構沒有改變,但是卻比較容易被蛋白酶Lys-C切割。此外,突變株並且出現新的喜好切割位。雖然hsc70(R447G)可以和CMLA形成穩定結合的複合體,但是複合體的量減少許多。hsc70(L399P)則是無法和CMLA形成穩定結合的複合體。以短胜肽FYQLALT測試各hsc70蛋白質和受質的作用發現,突變株釋出FYQLALT的速率比較快。在測試的hsc70和突變株之中,hsc70(R447G)的釋出速率最快。雖然突變株可以重新折疊鹽酸胍(guanidine hydrochloride)處理後變性的luciferase,但是hsc70(L399P)和hsc70(R447G)重新折疊活性分別降低為hsc70的40%和20%。這些結果顯示突變株的重新折疊去活蛋白的活性和受質解離速率之間有關連性。

It has been known that 70-kDa heat shock cognate proteins (hsc70) is capable of forming complexes with unfolded polypeptide substrates and works with DnaJ homologues to refold denatured proteins. Using native gel electrophoresis, I have showed that the 30-kDa C-terminal fragment forms tightly associated complexes with bovine S-carboxymethyl--lactalbumin (CMLA), indicating that the 30-kDa domain is responsible for complexing with unfolded proteins.
I also have characterized the substrate binding and refolding activities of two hsc70 mutants, hsc70(L399P) and hsc70(R447G). Both Leu-399 and Arg-447 are highly conserved among hsc70s in different organisms. The secondary structure of hsc70(L399P) and hsc70(R447G) remains the same as that of hsc70, but the mutants are more accessible for Lys-C digestion and also show new preferred cleavage sites. While hsc70(R447G) is capable of forming tightly associated complexes with CMLA, the level of the complexes is greatly reduced. Hsc70(L399P) however does not form such complexes with S-carboxy--lactalbumin. The heptapeptide FYQLALT also has been used to examine the interaction of hsc70 proteins with the substrates. FYQLALT is more readily to dissociate from the mutants. Among the hsc70 protein species examined, hsc70(R447G) has the largest dissociation rate constant. Moreover, the mutants retain the capability of restoring the activity of denatured luciferase, but the refolding activity of hsc70(L399P) and hsc70(R447G) has been decreased to 40% and 20% of that for hsc70, respectively. Taking together, the results indicate that the ability of these mutants to refold denatured proteins is correlated with the rate of substrate dissociation.

中文摘要 1
英文摘要 2
第一章 緒論 4
第一節 蛋白質的折疊與熱休克蛋白 4
第二節 hsc70是hsp70家族的成員 5
第三節 hsc70的折疊監護活性 6
第四節 hsc70作用的循環 7
第五節 hsc70 與受質的結合 9
第二章 hsc70碳端30k-Da片段和非折疊蛋白質受質緊密結合 11
第一節 簡介 12
第二節 材料與方法 13
第三節 結果 18
第四節 討論 21
第三章 hsc70突變株的分析 24
第一節 簡介 25
第二節 材料與方法 26
第三節 結果 31
第四節 討論 40
第四章 hsc70碳端30k-Da片段和TPR1的交互作用 44
第一節 簡介 45
第二節 材料與方法 46
第三節 結果 50
第四節 討論 52
第五章 參考文獻 56
表目錄 頁
表一、hsc70蛋白的水解酶活性 63
圖目錄 頁
圖說明 64
圖一 hsc70的結構 72
圖二 hsc70和CMLA形成複合體 73
圖三 hsc70碳端30-kDa和CMLA形成複合體 74
圖四 6xHis標籤不影響複合體的形成 75
圖五 hsc70作用的循環 76
圖六 大白鼠hsc70碳端30-kDa區間的結構模型 77
圖七 純化的hsc70蛋白 78
圖八 蛋白酶切割探測hsc70蛋白質的結構 79
圖九 CD光譜 80
圖十 hsc70蛋白質和CMLA形成的複合體 81
圖十一 CMLA濃度對複合體形成的函數關係 82
圖十二 試管中luciferase重新折疊 83
圖十三 結合胜肽由hsc70蛋白質釋出 84
圖十四 各hsc70蛋白質的重新折疊活性和受質解離速率的關連性 85
圖十五 hsc70或hsc70碳端30-kDa區間片段和TPR1在試管中結合 86
圖十六 TPR1的TPR模組區間和hsc70碳端30-kDa區間片段的交互作用 87
圖十七 TPR1抑制hsc70對luciferase重新折疊的監護活性 88
附錄 已發表論文 88

劉福華 (2000) 七萬道爾吞熱休克蛋白(hsc70)作用蛋白之篩選與分析。國防醫學院生命科學研究所博士論文。
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