高鹽甲烷太古生物ClpB蛋白之特性分析

Abstract

分子伴護蛋白ClpB屬於利用ATP來行使其功能的AAA+家族，具有將細胞內因逆境而形成的聚集蛋白，與DnaK分子伴護蛋白系統合作進行去聚集化反應使蛋白回復正常功能，進而保護細胞不受逆境影響而死亡。ClpB單體結構具有兩個核苷酸結合位，在與ATP結合時會形成環狀六聚體結構。ClpB除存在於真細菌與真核生物中，也在太古生物中發現到，其中於高鹽甲烷太古生物Methanohalophilus portucalensis FDF1中發現的分子伴護蛋白MpClpB，其轉錄表現會受到鹽逆境與熱逆境的誘導。本研究將MpClpB表現於clpB基因缺陷的E. coli BB4561中，顯示在高鹽逆境(1.0 M NaCl)下MpClpB的伴護功能較EcClpB佳，且明顯的縮短遲滯期。同時在E. coli BB4561中表現的MpClpB在高鹽環境下，亦具有去聚集蛋白的能力。因此，MpClpB的表現可能有助於避免高鹽逆境下新生蛋白的聚集，幫助細胞快速適應高鹽環境並生長。純化的異源表現MpClpB和EcClpB一樣也具有形成六聚體的能力，但是在相同測試條件下MpClpB的ATP水解量非常低僅至EcClpB的5%。但高濃度的蛋白與額外添加相容質betaine (0.6 M)，能提高MpClpB的ATP水解酶活性7倍，達到EcClpB ATP水解酶活性的40%活性，且在不同反應溫度與不同離子濃度顯現出與EcClpB相似的特性。本研究的結果確認高鹽甲烷太古生物的ClpB，具有ATP水解酶活性與形成六聚體和去聚集蛋白的能力，確為分子伴護蛋白。同時發現相容質betaine的濃度會影響與調節MpClpB的ATP水解酶活性。

Molecular chaperone ClpB belongs to the members of AAA+ protein superfamily, it could prevent the cell death from stress by mediating the disaggregation of the insoluble protein in cooperation with DnaK chaperone system. ClpB contains two nucleotide bind sites and forms the hexameric ring while binding ATP. ClpB is found in bacteria and eukaryotes but also found in archaea. ClpB also called MpClpB, found in the halophilic methanoarchaeon Methanohalophilus portucalensis FDF1, was investigated that the transcriptional level could be induced by the salt shock and heat shock. In this study, MpClpB was heterogenous expressed in clpB-null mutant E. coli BB4561 under high salt stress (1.0 M NaCl). The result shows that chaperone acitivity of MpClpB could reduce the lag time of E. coli BB4561 than EcClpB could do, and also prevent the aggregated protein accumulation under salt strss. It suggests that MpClpB could assist the E. coli BB4561 in growing at the high salt environment. The purified MpClpB could assemble into hexamer as EcClpB, but has low ATPase activity only reached the 5% ATPase activity of EcClpB. Nevertheless the ATPase activity of MpClpB was enhanced with the increased protein concentration and the addition of osmolyte betaine. With 0.6 M betaine the activity of MpClpB increased 7 folds and reached the 40% activity of EcClpB. Furthermore, the ATPase of MpClpB shows the same qualities as EcClpB investigated by previous studies under the different temparatures and ionic strengh. This study confirms that halophilic methanoarchaeon ClpB possesses chaperone characters including the ATPase activity, oligomerization and while cell suffering the salt stress. In addition, the osmolyte betaine plays an important role in regulating the ATPase activity of MpClpB.