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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer.
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HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer.

机译：日本根瘤菌根瘤菌的HypB蛋白是一种金属结合GTP酶，每个二聚体能够结合18个二价镍离子。

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摘要

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+. In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column. The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.
机译：日本慢生根瘤菌hypB编码一种蛋白质，该蛋白质包含一个非常富含组氨酸的区域（一个39个氨基酸的片段中有24个组氨酸残基）和鸟嘌呤核苷酸结合域。 hypB基因的产物在大肠杆菌中过表达，并通过一步一步通过带Ni（2+）的金属螯合亲和色谱（MCAC）进行纯化。在SDS / PAGE中，HypB以38 kDa迁移-略大于计算的分子量（32.8 kDa）。纯化的HypB具有GTPase活性，kcat为0.18 min-1，GTP的Km为7 microM，并且还具有dGTPase活性。通过在Superose 12上通过快速蛋白质液相色谱法测定，HypB在天然溶液中以分子量为78 kDa的二聚体形式存在。它与每个单体结合9.0 +/- 0.14二价镍离子（每个二聚体18 Ni2 +），Kd为2.3 microM。它还结合Zn2 +，Cu2 +，Co2 +，Cd2 +和Mn2 +。框内缺失富含组氨酸的区域（删除38个氨基酸，包括23个组氨酸残基）导致截短的HypB不与MCAC柱结合，而框内缺失14个氨基酸，包括HypB中的8个组氨酸残基导致截断的HypB仍然绑定到该列。结果表明，HypB富含组氨酸的区域内的组氨酸残基参与金属结合。

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