精胺酸定點突變對嗜熱短桿菌Lon蛋白水解酵素熱穩定度及功能之影響

Effects of Arginine mutations on functions and thermostability of Lon protease from brevibacillus thermoruber

黃金源(Chin-Yuan Huang)

指導教授 ： 吳世雄

國立臺灣大學/生命科學院/生化科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02386

摘要

維持蛋白質弁鉬P結構的恆定對生物細胞正常運作是相當重要的。在細胞內，透過蛋白酶分解可去除弁鄐庰硎c上不正常的蛋白並且控制一些調控性蛋白的運作。細胞內的蛋白質分解主要是藉由一種ATP依賴性寡聚蛋白水解酶(ATP-dependent oligomeric protease)所執行。在大腸桿菌中，目前已發現的ATP依賴性蛋白水解酶共有五種，分別為：Lon，FtsH，ClpAP，ClpXP，與HslUV。
在實驗室先前研究中，已從台灣烏來溫泉菌-嗜熱短桿菌(Brevibacillus thermoruber WR-249)中選殖出Lon蛋白酶(Bt-Lon)，並對其作生化鑑定。在先前研究中，我們發現到Bt-Lon的鹼性氨基酸組成中含有較高比例的精胺酸 (arginine)。我們認為此現象可能與Bt-Lon的DNA結合活性及熱穩定相關。因此在本論文中，我們從Bt-Lon選出七個分佈於不同弁鈰洈犖邆i酸，並利用單點突變來探討它們所扮演的弁鄔囧丹漶A它們分別為Arg98，Arg370，Arg518，Arg557，Arg566，Arg590，與Arg697。
由圓二色偏光儀及螢光儀光譜顯示，Bt-Lon的二級及三級結構並未受到上述七個精胺酸單點突變的影響而改變。由熱變性實驗的變性曲線可知，所有的突變型Bt-Lon的Tm值與野生型相近，均約為71.5 °C。換言之，由丙胺酸取代上述精胺酸的單點突變並不影響Bt-Lon的熱穩定度。
在酵素活性方面，所有突變型Bt-Lon均具有與野生型類似的蛋白水解酶及ATP水解酶活性。在DNA結合活性方面，R518A，R557A，與R566A這三個突變型蛋白的DNA結合力則顯著降低。更進一步，我們發現到DNA無法活化R518A與R566A兩個突變型蛋白的酵素活性。由Bt-Lon的一級結構得知，Arg518與Arg566皆位於受質偵測識別弁鈰?sensor- and substrate- discrimination domain, SSD domain)。而Bt-Lon的SSD弁鈰洃w被證實具有DNA結合弁遄C在Bt-SSD弁鈰洈犒q腦模擬結構中，含有一個高密度正電凹面區，而Arg518與Arg566正好位在此區內。以上結果顯示，這個高密度正電凹面區可能就是Bt-Lon的DNA結合區，而位於此區的Arg518與Arg566可能直接參與Bt-Lon與DNA的結合。

關鍵字

嗜熱短桿菌 ； 受質偵測識別弁鈰?聚合鍊鎖反應

並列摘要

Proteolysis in living cells serves to rid the abnormal and misfolded proteins of the cell and to limit the time and amounts of the availability of critical regulatory proteins. Most intracellular proteolysis is carried out by oligomeric ATP-dependent proteases containing both ATPase and proteolytic activities. Five ATP-dependent proteases, Lon, FtsH, ClpAP, ClpXP, and HslUV, have been discovered in Escherichia coli. Among them, Lon protease has been found in virtually all living organisms, from archaea to eubacteria to humans.
A thermostable Lon protease from thermophilic Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized in our laboratory. Previous studies showed that this protease possesses a higher ratio of arginine, and these residues may be involved in DNA-binding activity as well as responsible for thermostability of Bt-Lon under high temperature. Here, seven arginine residues of Bt-Lon, Arg98, Arg370, Arg518, Arg557, Arg566, Arg590, and Arg697, were selected for point-mutagenic analysis to investigate their functional roles.
The overall structure of each mutant determined by CD spectrum and fluorescence spectroscopy was not affected by the site-specific point mutations. Furthermore, all mutants showed a Tm value of 71.5 °C, identical to that of wild-type Bt-Lon. These results indicated that the substitution of Arg98, Arg370, Arg518, Arg557, Arg566, Arg590, or Arg697 with alanine did not affect the thermostability of the proteins.
All mutants displayed the basal peptidase and ATPase activities of the wild-type. Nevertheless, the three mutants, R518A, R557A, and R566A, showed a lower DNA-binding affinity based on the GMSA experiment. Furthermore, the two mutants, R518A and R566A, exhibited their peptidase and ATPase activities which are not stimulated by DNA. The results suggested that the DNA-binding affinity for Bt-Lon directly influences the activities of peptidase and ATPase. Based on the modeling structure of Bt-SSD, the two residues, Arg518 and Arg566, were located in the highly positively-charged surface of SSD domain that is proved to be a DNA-binding domain of Bt-Lon. In summary, our results indicated that Arg518 and Arg566, which are located in the highly positively-charged surface of SSD domain, are directly involved in DNA binding.

並列關鍵字

AAA+ ； CD ； PCR ； GMSA ； SSD domain ； Bs-Lon ； Bt-Lon

參考文獻

1. Gottesman, S., Maurizi, M. R. & Wickner, S. (1997) Regulatory subunits of energy-dependent proteases, Cell. 91, 435-8.

2. Charette, M. F., Henderson, G. W. & Markovitz, A. (1981) ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12, Proc Natl Acad Sci U S A. 78, 4728-32.

3. Chung, C. H. & Goldberg, A. L. (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La, Proc Natl Acad Sci U S A. 78, 4931-5.

4. Swamy, K. H. & Goldberg, A. L. (1981) E. coli contains eight soluble proteolytic activities, one being ATP dependent, Nature. 292, 652-4.

6. Goldberg, A. L., Moerschell, R. P., Chung, C. H. & Maurizi, M. R. (1994) ATP-dependent protease La (lon) from Escherichia coli, Methods Enzymol. 244, 350-75.