水稻小分子量熱休克蛋白質Oshsp16.9A之N端區域功能性分析

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李易諠(Yi-Hsuan Lee) 查詢紙本館藏

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水稻小分子量熱休克蛋白質Oshsp16.9A之N端區域功能性分析
(Functional study of N-terminal domain of a rice small heat shock protein, Oshsp16.9A)

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摘要(中)

小分子量熱休克蛋白質(Small heat shock proteins; sHSPs)可以分成七群，主要生理功能在熱逆境下避免不正常的蛋白質聚集，具不需要消耗能量(ATP)的chaperone活性。水稻小分子量熱休克蛋白質Oshsp16.9A第二保留區的第73和74個胺基酸已經被證實為與substrate產生交互作用的位置，去除此區域時會造成chaperone活性喪失。本篇論文，主要在了解Oshsp16.9A的N端區域在chaperone活性上所扮演的角色。因此，建構了N端區域刪除的Oshsp16.9A片段，利用大腸桿菌表現，經純化後進行生理功能試驗分析。結果顯示N端區域刪除的Oshsp16.9A片段在形成蛋白質複合體(protein complex)上發生困難，去除N端29個以上的胺基酸時會喪失chaperone活性，若去除N端35個以上的胺基酸時，會造成oligomers不穩定。雖然Oshsp16.9AN11-150(刪除N端第一至第十個胺基酸區域的Oshsp16.9A)無法形成正常的蛋白質複合體(protein complex)，但仍具有Oshsp16.9AFL一樣的chaperone活性，因此推測N端第一至第十個胺基酸區域在suboligomers間的相互作用上，扮演重要的角色；此外，試管內(in vitro)功能分析顯示第11到29個胺基酸區域在幫助suboligomers提供chaperone活性時是很重要的；根據Glutaraldehyde cross-link結果得知，第30到35個胺基酸區域是負責穩定suboligomers的形成。雖然Oshsp16.9AFL和Oshsp16.9AN11-150在試管內實驗都具有chaperone活性，但在活體(in vivo)實驗中，卻無法增加耐熱性。然而，大量表現Oshsp16.9AN30-150(刪除N端第一至第十個胺基酸區域的Oshsp16.9A), Oshsp16.9AN37-150(刪除N端第一至第三十六個胺基酸區域的Oshsp16.9A), Oshsp16.9AN43-150(刪除N端第一至第四十二個胺基酸區域的Oshsp16.9A)時卻比沒有蛋白質表現時，在高溫情況下造成更多的死亡。因此，我們推測大量表現Oshsp16.9A對於大腸桿菌菌種BL21 (DE3)的耐熱性，不會提供有效地幫助。此外，N端區域缺失的突變Oshsp16.9A大量的表現將會干擾大腸桿菌菌種BL21 (DE3)天生的耐熱性。

摘要(英)

Small heat shock proteins (sHSPs) can be divided into seven classes and exhibit ATP-independent molecular chaperone activity that can prevent denaturing proteins from aggregation. Rice (Oryza sativa) class I small heat shock protein, Oshsp16.9A, has been shown to be able to confer thermotolerance in Escherichia coli. The deletion of amino acid residues 73 and 74 in the consensus II domain of Oshsp16.9A led to loss of chaperone activity. Here we try to understand the role of the N-terminus of Oshsp16.9A in chaperone activity. Therefore, the N-terminus deletion mutants of Oshsp16.9A were constructed and overexpressed in E. coli cells for analyzing protein function. We found that the N-terminus deletion mutants of Oshsp16.9A could not form protein complex as Oshsp16.9A did. The deletion of N-terminal 1 through 29 amino acid residues of Oshsp16.9A led to the loss of chaperone activity. Besides, the deletion of N-terminal 1 through 35 amino acid residues of Oshsp16.9A caused oligomer unstable. Although Oshsp16.9AN11-150 could not form similar size of protein complex as Oshsp16.9AFL did, Oshsp16.9AN11-150 still had chaperone activity as Oshsp16.9AFL did. Therefore, it is suggested that the region of 1st to 10th amino acid of Oshsp16.9A plays an important role in suboligomer-suboligomer interaction. According to the analysis of in vitro chaperone activity, the region of 11th to 29th amino acid of Oshsp16.9A is important for its chaperone activity. Moreover, using glutaraldehyde cross-linking, the function of the region of 30 through 35 amino acid of Oshsp16.9A was found stabilize formation of suboligomers. Although Oshsp16.9AFL and Oshsp16.9AN11-150 have chaperone activity in vitro, they did not confer thermoprotection in E. coli. On the other hand, overexpression of Oshsp16.9AN30-150, Oshsp16.9AN37- 150, and Oshsp16.9AN43-150 caused lower survival rates than that of control under high temperature. Therefore, we suggested that overexpression of Oshsp16.9A did not help thermotolerance efficiently in E. coli strain BL21 (DE3). Furthermore, overexpression of Oshsp16.9A N-terminal mutants would interfere with natural thermotolerance of E. coli strain BL21 (DE3).

關鍵字(中)

★ 小分子量熱休克蛋白質
★ N端區域

關鍵字(英)

★ heat
★ small heat shock protein
★ N-terminal region
★ hsp16.9A

論文目次

Chinese abstract－－－－－－－－－－－－－－－－－－－－－－－－－－－－－I
Abstract－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－II
Contents－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－III
List of abbreviations－－－－－－－－－－－－－－－－－－－－－－－－－－VI
Chapter 1. Introduction
1.1 The heat stress－－－－－－－－－－－－－－－－－－－－－－－－－－－－1
1.2 Classification of HSPs and Property of HSPs－－－－－－－－－－－－－－－－1
1.3 Classification of Plant sHSP－－－－－－－－－－－－－－－－－－－－－－－2
1.4 Structure of sHSPs－－－－－－－－－－－－－－－－－－－－－－－－－－－3
1.5 Interaction Region of sHSPs－－－－－－－－－－－－－－－－－－－－－－－4
1.6 History of Rice Oshsp16.9A－－－－－－－－－－－－－－－－－－－－－－－5
1.7 Specific Aim－－－－－－－－－－－－－－－－－－－－－－－－－－－－－6
Chapter 2. Materials and Methods
2.1 Preparation of Oshsp16.9A Expression Constructs：
(A) PCR (Polymerase Chain Reaction) －－－－－－－－－－－－－－－－－－－7
(B) Extraction of Plasmid DNA －－－－－－－－－－－－－－－－－－－－－－7
(C) Digestion－－－－－－－－－－－－－－－－－－－－－－－－－－－－－8
(D) Gel Elution－－－－－－－－－－－－－－－－－－－－－－－－－－－－8
(E) Ligation－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－9
2.2 Expression and Purification of the OsHSPs－－－－－－－－－－－－－－－－10
2.3 Preparation of PAGE－－－－－－－－－－－－－－－－－－－－－－－－－10
2.4 Native PAGE Analysis－－－－－－－－－－－－－－－－－－－－－－－－12
2.5 Glutaraldehyde Cross-link－－－－－－－－－－－－－－－－－－－－－－－12
2.6 Western Blotting－－－－－－－－－－－－－－－－－－－－－－－－－－－13
2.7 Size Exclusion Chromatography (SEC) of the OsHSPs－－－－－－－－－－13
2.8 Assays of Chaperone Activity－－－－－－－－－－－－－－－－－－－－－14
2.9 Thermotolerance Assays for Transformants－－－－－－－－－－－－－－－－14
Chapter 3. Results
3.1 Construction and Expression of Oshsp16.9A Variants－－－－－－－－－－－－16
(A) Induction of Oshsp16.9A variants in E. coli strain BLR (DE3)－－－－－－－－16
(B) Purification of Oshsp16.9A variants－－－－－－－－－－－－－－－－－－17
3.2 Conformation Analysis of Oshsp16.9A Variants－－－－－－－－－－－－－－－17
(A) Analysis of the native complexes of Oshsp16.9A variants using native PAGE－－17
(B) Characterization of oligomerization of Oshsp16.9A variants using glutaraldehyde----
cross-link－－－－－－－－－－－－－－－－－－－－－－－－－－－－－17
(C) Study of the sizes of complexes of Oshsp16.9A variants using SEC－－－－－－18
3.3 Activity Assay of Oshsp16.9A Variant in Vitro－－－－－－－－－－－－－－18
(A) Chaperone activity of Oshsp16.9A variants－－－－－－－－－－－－－－－18
(B) Binding substrate activity of Oshsp16.9A variants－－－－－－－－－－－－18
3.4 Assay of Thermotolerance of E. coli Expressing Oshsp16.9A Variants in Vivo－－－19
(A) Thermotolerance of E. coli expressing Oshsp16.9A variants－－－－－－－－－19
(B) The effects of E. coli expressing Oshsp16.9A variants at high temperature－－－19
Chapter 4. Discussion
4.1 N-terminus of Oshsp16.9A plays an important role in oligomerization－－－－－－20
4.2 N-terminal

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指導教授

葉靖輝(Ching-Hui Yeh)

審核日期

2006-11-25

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