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研究生:吳至忠
研究生(外文):Chih-Chuang Wu
論文名稱:Hsp70 伴護蛋白系統之結晶學研究: 熱休克蛋白DnaK 與其協同伴護蛋白GrpE 間的分子交互作用
論文名稱(外文):Crystallographic Studies of the Hsp70 Chaperone System: Insight into the DnaK and GrpE Intermolecular Interaction
指導教授:蕭傳鐙蕭傳鐙引用關係簡靜香簡靜香引用關係
指導教授(外文):Chwan-Deng HsiaoChin-Hsiang Chien
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:63
中文關鍵詞:熱休克蛋白Hsp70協同伴護蛋白GrpE伴護蛋白互補分析
外文關鍵詞:Hsp70GrpEChaperoneComplementation assay
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Hsp70 伴護蛋白系統之結晶學研究: 熱休克蛋白DnaK 與其協同伴護蛋白GrpE 間的分子交互作用

摘要
熱休克蛋白Hsp70 是一種存在大多數生物體內具有高度保留性的分子伴護蛋白分子,其主要功能是維持細胞內其他蛋白質的正常折疊。蛋白質DnaK 是Hsp70 在原核物種內的同源蛋白質。在原核細胞內,DnaK 與其協同伴護蛋白分子DnaJ 與GrpE 共同運作來幫助其他蛋白質摺疊。然而由於缺乏詳細結構資訊,我們無法清楚了解上述分子彼此間的作用機制。在本研究裡,我們成功從嗜熱性土壤芽孢桿菌(Geobacillus kaustophilus HTA426)內解出了由全長協同伴護蛋白GrpE 與近乎全長(包含氨端的核苷酸結合結構域, 羧端的受質結合結構域以及連接這兩個結構域的多肽鍊)的伴護蛋白DnaK 所形成蛋白質複合體的三維空間立體結構。結構內發現每個複合體都由兩個DnaK分子與兩個GrpE 分子結合而成,形成之前所沒發現過的結合比例。結構中發現在形成複合體後GrpE 分子其氨端的α-螺旋二級結構可以穩定DnaK 分子內的多肽鍊區域。另外我們也發現形成複合體後位於DnaK 分子羧端的受質結合結構域與GrpE 分子氨端的不規則胺基酸鍊區域間的結合有可能促進DanK 釋放原來結合住的多肽受質。本篇研究的發現對於熱休克蛋白Hsp70 伴護分子系統(Hsp70 chaperone system)中的受質結合,處理與釋放等步驟的機制都提供了進一步的了解。
Crystallographic Studies of the Hsp70 Chaperone System: Insight into the DnaK and GrpE
Intermolecular Interaction

Abstract
The 70-kDa heat shock proteins (Hsp70s) are highly conserved molecular chaperones composed of an N-terminal nucleotide binding domain (NBD) and a C-terminal substrate binding domain (SBD) in a bilobate mode. In prokaryotic cell,The conserved, ATP-dependent bacterial DnaK chaperones (Hsp70 homologous)process client substrates with the aid of the co-chaperones DnaJ and GrpE, this DnaK chaperone machine is usually termed DnaK chaperone cycle. In this cycle, the GrpE function as a nucleotide exchange factor to promote dissociation of ADP from nucleotide-binding cleft of DnaK. However, in the absence of structural information,how DnaK and GrpE communicate with each other cannot be fully delineated. For the study reported herein, we solved the crystal structure of a full-length Geobacillus
kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the N-terminal binding domain, interdomain linker and C-terminal substrate binding domain at 4.1-Å resolution. The GkDnaK-GkGrpE complex structure contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex and this interaction was crucial for in vivo function of DnaK. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substratebinding, processing, and release during the Hsp70 chaperone cycle.
Contents
Chinese abstract ---------------------------------------------------------------------I
English abstract --------------------------------------------------------------------II
Contents ----------------------------------------------------------------------------IV
List of Tables ----------------------------------------------------------------------VI
List of Figures --------------------------------------------------------------------VII
List of Abbreviations ---------------------------------------------------------------IX

Chapter 1 Introduction ---------------------------------------------------------------1

Chapter 2 Materials and Methods ------------------------------------------------------5
2.1 Cloning, Protein Expression and Purification -------------------------------------5
2.2 GkDnaK-GkGrpE Complex Preparation ------------------------------------------------6
2.3 Crystallization of the GkDnaK-GkGrpE Complex -------------------------------------7
2.4 Data Collection, Structure Determination and Refinement --------------------------8
2.5 Isothermal Titration Calorimetry -------------------------------------------------9
2.6 In Vivo Complementation Assay ----------------------------------------------------9

Chapter 3 Results -------------------------------------------------------------------11
3.1 Overall Structure of the GkDnaK-GkGrpE Complex ----------------------------------11
3.2 Stoichiometry of DnaK and GrpE --------------------------------------------------12
3.3 Flexible DnaK Interdomain Linker Allows Dynamic Motion of DnaK_SBD --------------13
3.4 Nucleotide-binding Pocket Widens upon GrpE Binding ------------------------------14
3.5 Similarities and Differences among GrpE Structures ------------------------------15
3.6 Long N-terminal α-Helices of GkGrpE Stabilize Ternary Complex -------------------16
3.7 In Vivo Complementation Assays for DnaK -----------------------------------------17

Chapter 4 Discussion ----------------------------------------------------------------19

Chapter 5 Future work ---------------------------------------------------------------23

Table -------------------------------------------------------------------------------24

Figures -----------------------------------------------------------------------------28

References --------------------------------------------------------------------------57
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