臺灣博碩士論文加值系統

許多分子伴護蛋白皆為熱休克蛋白 (Hsp) ，且在保護細胞對抗環境中許多逆境扮演著重要的角色。DnaK系統被大家所知其功能在於幫助新合成蛋白摺疊及避免細胞內蛋白質聚集，且具有兩個輔助性分子伴護蛋白DanJ與GrpE，能夠提高DnaK之活性。
在本研究中，分為兩個部分探討Bacillus licheniformis ATCC 14580熱休克蛋白70 (BlDnaK)系統中，成員DnaK與GrpE之特定殘基對其結構與活性之影響：
PCR的過程中， 我們得到了在第52及134號密碼子雙點突變之BlGrpE突變株。BlGrpE之殘基Leu52與Leu134分別以脯胺酸和組胺酸取代，為兩突變株BlGrpE-L52P與BlGrpE-L134H。BlGrpE野生型與BlGrpE-L52P皆能促進BlDnaK三磷酸腺苷水解活性。然而BlGrpE-L134H與雙點突變之BlGrpE-L52P/L134H並不具有輔助性分子伴護蛋白之能力。BlGrpE野生型、BlGrpE-L52P、BlGrpE-L134H能與BlDnaK單體進行交互作用，但BlGrpE-L52P/L134H未發現具有專一性交互作用。測量螢光光譜發現BlGrpE突變株之芳香族胺基酸殘基所處之微環境發生了重大的變化。與BlGrpE野生型相比，BlGrpE突變株蛋白之圓二色光譜於波長208 nm與222 nm之訊號減少，且對於溫度所引起之變性作用更加敏感。
為了進一步探討BlGrpE第134個胺基酸殘基所扮演的角色，因此進行飽和選位突變。利用鎳金屬螯合層析管柱純化BlGrpE野生型及其突變株蛋白質，且其分子量約為34.5 kDa。BlGrpE野生型進行核苷酸交換因子之活性比較時，可以發現其突變株L134H、L134K、L134R、L134D、L134E、L134N、L134Q、L134S、L134G與L134P之活性減少超過96 %。在試管中進行BlDnaK與BlGrpE之結合分析實驗，顯示BlGrpE野生型與具有功能之突變株皆能與BlDnaK單體進行交互作用，而剩下的突變株則無法與BlDnaK單體進行交互作用。BlGrpE野生型與其他九個具有功能性之突變株可增加刺激BlDnaK之三磷酸腺苷水解活性，而其他缺陷之突變株則否。依據圓二色光譜之比對分析後，發現不具活性之突變株螺旋含量明顯少於野生型。此外，不具活性之突變株對於溫度所引起之變性作用更為敏感。綜合上述結果，說明BlGrpE殘基Leu134可能在結構上扮演著重要的角色。
DnaK在三磷酸腺苷水解反應過程中，需特定胺基酸殘基維持DnaK之三磷酸腺苷鍵結區中ADP-Mg-Pi複合物處於一正確構型。透過序列同質性，我們模擬出BlDnaK 之三維結構，並選出五個可能與維持此構型有關之胺基酸殘基。與BlDnaK野生型相較之下，選位突變之蛋白質D8A、N13D、E145A、D168A與T173A，其分子伴護蛋白活性劇烈地下降。補償性實驗中顯示突變蛋白質完全喪失復活大腸桿菌 (Escherichia coli) dnaK756-ts熱敏感生長缺失突變株之生長能力。BlDnaK野生型能夠幫助變性之螢火蟲螢光酵素再折疊，然而突變株蛋白質此能力則是大幅度下降。同時加入BlDnaJ、BlGrpE及NR-胜肽，D8A、N13D、E145A、D168A與T173A之三磷酸腺苷水解活性並無協同刺激。除D8A外，野生型與其餘突變株蛋白質皆有幾乎相同之圓二色光譜，且對溫度誘導變性具有相似之敏感性。這些結果暗示選擇之殘基對BlDnaK之功能性非常重要。

Many molecular chaperones are heat-shock proteins (Hsp) and play an important role in the protection of the host cell against various stresses. DnaK system is a known for assisting protein folding and avoiding intracellular protein aggregation, it has two co-chaperones DnaJ and GrpE, they could accelerate the DnaK activity.
In this study two researches are conducted to invesigate the effect of specific residues of DnaK and GrpE for structural and chaperone activity in Bacillus licheniformis ATCC 14580 heat shock protein 70 (BlDnaK) system.
A DNA fragment encoding BlGrpE with double mutations at codons 52 and 134 was obtained during PCR cloning. Leu52 and Leu134 in BlGrpE were individually replaced with Pro and His to generate BlGrpE-L52P and BlGrpE-L134H. BlGrpE and BlGrpE-L52P synergistically stimulated the ATPase activity of BlDnaK. However, BlGrpE-L134H and the double-mutated protein (BlGrpE-L52P/L134H) had no co-chaperone function. BlGrpE, BlGrpE-L52P, and BlGrpE-L134H mainly interacted with the monomer of BlDnaK but non-specific interaction was observed for BlGrpE-L52P/L134H. Measurement of intrinsic fluorescence revealed a significant alteration of the microenvironment of aromatic acid residues in the mutant proteins. As compared with BlGrpE, quenching of 208 nm and 222 nm signals were observed in the mutant BlGrpEs and the single-mutated proteins were more sensitive to thermal denaturation.
To further elucidate the role of leucine 134 of BlGrpE, site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of BlDnaK, whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the a-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.
DnaK needs specific residues maintaining ADP-Mg2+-Pi complex at the ATP-binding cleft of protein in ATP hydrolysis. Based on the sequence homology, we have modeled the three-dimensional structure of BlDnaK, and selected five different amino acids that might be responsible for the complex. As compared with wild-type BlDnaK, site-directed mutant proteins D8A, N13D, E145A, D168A, and T173A had a dramatic reduction in their chaperone activities. Complementation test revealed that the mutant proteins lost completely the ability to rescue the temperature-sensitive growth defect of Escherichia coli dnaK756-ts. Wild-type BlDnak assisted the refolding of denatured firefly luciferase, whereas a significant decrease in this ability was observed for the mutant proteins. Simultaneous addition of BlDnaJ, BlGrpE, and NR-peptide, did not synergistically stimulate the ATPase activity of D8A, E145A, D168A and T173A. Circular dichroism spectra were nearly identical for wild-type and mutant proteins, and they, except D8A, also exhibited a similar sensitivity towards temperature-induced denaturation. These results suggest that the selected residues are critical for the proper function of BlDnaK.

英文摘要 1
中文摘要 4
前言
一、細胞逆境反應 7
二、熱休克蛋白 9
三、熱休克蛋白70 13
四、熱休克蛋白40 23
五、核苷酸交換因子 GrpE 26
六、原核生物中熱休克蛋白70之機制 29
七、螢光光譜及圓二色光譜 33
八、Bacillus licheniformis ATCC14580 38
九、目前所要進行之研究 41
材料與方法
一、藥品 45
二、菌株質體及培養基 45
三、方法 47
I. BlGrpE實驗方法與步驟
1. 質體 47
2. 重組DNA技術 47
3. 選位突變 54
4. 少量抽取BlGrpE質體及轉型至大腸桿菌M15 58
5. BlGrpE 之表現及純化 60
6. 蛋白質電泳分析與活性染色 63
7. 酵素之生化特性分析 68
8. 核苷酸釋放量 70
9. BlGrpE 之光譜結構特性分析 72
II. BlDnaK實驗方法與步驟
1. 質體 73
2. 選位突變 74
3. 少量抽取BlDnaK質體及轉型至大腸桿菌M15 74
4. BlDnaK 之表現及純化 75
5. 蛋白質電泳分析與活性染色 78
6. 酵素之生化特性分析 80
7. BlDnaK動力學之分析 85
8. 螢光酵素復性分析 91
9. BlDnaK野生型及其突變株之光譜結構特性分析 91

結果
一、 Bacillus licheniformins之核苷酸交換因子GrpE其殘基Leu52與Leu134對其整體結構而言相當重要 93
1. 序列比對與蛋白質純化 93
2. 純化之BlGrpEs其輔助性分子伴護蛋白活性與DnaK鍵
結之能力分析 96
3. 光譜特性 99
二、B. licheniformis核苷酸交換因子GrpE其殘基Leucine134飽
和選位突變之探討顯示此殘基對輔助性蛋白活性之重要性 104
1. BlGrpE與EcGrpE之序列比對 104
2. 選位飽和突變及其重組蛋白質之純化 106
3. BlGrpE野生型與其突變株之功能性分析 107
4. BlGrpE野生型及其突變株與DnaK鍵結活性分析 107
5. 純化之BlGrpE野生型及其突變株之輔助性分子伴護蛋
白活性 107
6. 結構分析 110
三、B. licheniformis DnaK突變株之分子伴護蛋白能力 114
1. BlDnaK之序列比對與同源模擬 114
2. 野生型及其突變株之特性分析 117
3. 大腸桿菌熱敏感突變株補償性分析 119
4. GdnHCl變性之螢火蟲螢光酵素之復性實驗分析 123
5. 輔助性分子伴護蛋白及胜肽對BlDnaK野生型與突變株之三磷酸腺苷水解活性影響 124
6. BlDnaK野生型與突變株之圓二色光譜探討 126
討論
一、 Bacillus licheniformins之核苷酸交換因子GrpE其殘基
Leu52與Leu134對其整體結構而言相當重要 131
二、 B. licheniformis核苷酸交換因GrpE其殘基Leucine134飽
和選位突變之探討顯示此殘基對輔助性蛋白活性之重要
性 137
三、 B. licheniformis DnaK突變株之分子伴護蛋白能力 142
參考文獻 149
附錄一 168
附錄二 169
附錄三 171
附錄四 172
附錄五 173
附錄六 174

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