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研究生:游約翔
研究生(外文):Yueh-hsiang Yu
論文名稱:綠豆Hsc70蛋白質與受質結合之專一性
論文名稱(外文):Substrate specificities of Hsc70s of Vigna radiata
指導教授:林彩雲林彩雲引用關係
指導教授(外文):Tsai-Yun Lin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
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:英文
論文頁數:58
中文關鍵詞:熱休克蛋白
外文關鍵詞:heat shock protein
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分子量70 kDa之熱休克同源蛋白(Hsc70)隸屬於分子監護子族群,為持續性於細胞內表現之監護子。其作用在於,避免處於高度擁擠的細胞環境下之蛋白質不正常堆疊及相互間不當聚合。在ATP的調控下,Hsc70會與共同監護子Hsp40合作,並且重複的結合與釋放新生成或非正常狀態下之蛋白質所暴露延展於外的月生 月太 鏈。而Hsc70對受質的專一性,在細菌、酵母菌、脊椎動物和植物有著不同的特性。為了要探討Hsc70和其受質間的交互作用及專一性,三個綠豆(Vigna radiata)Hsc70的C端30 kDa部分已大量表現,且用以當作目標蛋白來進行噬菌體表現篩選,進而篩選出可和VrHsc70結合之七子胺基酸鏈。三個出現頻率最高的七子胺基酸鏈對應其目標蛋白為:KVWVLPI對應於VrHsc70-1、KLWVIPQ對應於VrHsc70-2、KLWVIPQ及YAPLSRL對應於VrHsc70-3。而VrHsc70和其受質胺基酸鏈的結合能力已再由ELISA分析加以應證。這三個七子胺基酸鏈也用來搜尋含有相似序列的蛋白質,我們的結果顯示VrHsc70-1可能主要作用於幫助新生成或非正常狀態下之蛋白質正確的堆疊,而VrHsc70-2及3可能主要在參與蛋白質新合成過程。為了更進一步的研究三個七子胺基酸鏈和VrHsc70間結合的型式,電腦軟體InsightII則用以進行模擬七子胺基酸鏈進駐VrHsc70的方式。而擁有最低結合能量的模型已檢視,VrHsc70和KVWVLPI、KLWVIPQ及YAPLSRL間的結合有著相似的模式。

The 70 kilodalton heat shock cognate protein (Hsc70) is a constitutively expressed member of the molecular chaperones which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Hsc70 cooperates with cochaperone Hsp40, and functions by binding and releasing the exposed extended polypeptide of nascent and nonnative proteins, in the ATP-dependent manner. Binding specificities of Hsc70s to their substrates are quite different among bacteria, yeast, vertebrate and plant. To investigate the interactions and specificity between Hsc70 and its substrates, the carboxyl-terminal 30 kDa of three mung bean (Vigna radiata) Hsc70s were expressed and used as target proteins to select Hsc70-binding heptapeptides using phage display screening. Three heptapeptides of high frequencies were selected with the target proteins: KVWVLPI for VrHsc70-1, KLWVIPQ for VrHsc70-2, KLWVIPQ and YAPLSRL for VrHsc70-3. The target binding was confirmed by ELISA analysis. These three heptapeptides were subject to search for the homologous proteins. Our results suggested that VrHsc70-1 may assist the nonnative or newly synthesized proteins to fold correctly, and VrHsc70-2 and 3 may bind substrates during de novo synthesis stages. To study the binding patterns of three significant heptapeptides to VrHsc70s, InsightII program was used to docking each heptapeptide into its target VrHsc70. The lowest-energy binding models were inspected and a similar binding mode of VrHsc70s to KVWVLPI, KLWVIPQ and YAPLSRL was obtained.

Abstract (in Chinese) i
Abstract ii
Acknowledgments iii
Table of Contents iv
List of Tables vi
List of Figures vii
Abbreviations viii
Introduction 1
Materials and Methods 6
Result 17
Discussion 24
References 29
Tables 34
Figures 46

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