Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions
Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions
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DOI：
10.1023/a:1006273124726
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发表时间：
1999-06-01
影响因子：
5.1
通讯作者：
Takabe, T
中科院分区：
文献类型：
--
作者：
Hibino, T;Kaku, N;Takabe, T
此前，人们发现耐盐蓝藻Aphanothece halophytica的dnaK1基因编码721个氨基酸的多肽，该多肽具有长的C端区域，富含酸性氨基酸残基。为了了解盐生植物 DnaK1 在高盐度下是否具有分子伴侣活性，并阐明额外的 C 端氨基酸的作用，一项比较研究检测了三种 DnaK 分子的 ATP 酶活性以及其他尿素变性蛋白在不同盐度条件下的重折叠活性。来自 A. halophytica 和聚球藻 sp. 的 DnaK1。 PCC 7942 和 C 端缺失的 A. halophytica DnaK1 在大肠杆菌中表达并纯化。即使在高盐度（1.0 M 氯化钠或氯化钾）下，A. halophytica DnaK1 的 ATP 酶活性也非常高，而聚球藻 PCC 7942 DnaK1 中的这种活性随着氯化钠或氯化钾浓度的增加而降低。 DnaK1s 对尿素变性乳酸脱氢酶重折叠活性的盐依赖性与相应 DnaK1s 的 ATP 酶活性相似。 A. halophytica DnaK1 C端氨基酸的缺失对ATPase活性没有影响，但导致其他变性蛋白的重折叠活性显着降低。这些事实表明，A. halophytica DnaK1 的额外 C 末端区域在高盐度下其他尿素变性蛋白的重折叠中发挥着重要作用。此外，研究表明，在这些铜蛋白的折叠过程中，DnaK1 可以协助前体脱辅基体蓝蛋白以及成熟脱辅基体蓝蛋白的铜结合。
Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To understand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity (1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl. The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK1 had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins. These facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins.