Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.
Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.
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通过有限的蛋白水解和定向诱变鉴定红藻 PSII 中外在 12 kDa 蛋白的功能域。
DOI：
10.1093/pcp/pce170
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发表时间：
2001
影响因子：
4.9
通讯作者：
I. Enami
中科院分区：
文献类型：
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作者：
A. Okumura;H. Ohta;Y. Inoue;I. Enami
红藻光系统 II (PSII) 中的外在 12 kDa 蛋白可最大限度地减少放氧活动对氯和钙的需求 [Enami 等人，2017]。 (1998)生物化学37：2787]。为了鉴定12 kDa蛋白的功能域，我们通过有限蛋白水解和定向诱变制备了缺乏N端肽或C端肽或两者的12 kDa蛋白。通过重构实验检查所得 12 kDa 蛋白质片段的结合和功能特性。 (1)通过V8蛋白酶制备从Gly-6到12kDa蛋白质C末端的肽片段。该片段完全反弹至PSII，并且在不存在Cl(-)和Ca(2+)离子的情况下部分地重新激活氧析出，但在存在Cl(-)离子的情况下显着地重新激活氧的析出。 (2)通过胰凝乳蛋白酶处理获得从Leu-10到Phe-83的肽。该肽有效地反弹至 PSII，但在存在和不存在 Cl(-) 和 Ca(2+) 离子的情况下，重新结合并不能恢复析氧。 (3)两种突变蛋白，一种缺失N端5个残基，另一种缺失N端9个残基，能够有效结合PSII。通过它们的结合释放的氧气的恢复几乎与用 V8 蛋白酶处理的肽重建的相同。 (4) C端缺少10个、7个或3个残基的三种突变蛋白有效地反弹至PSII，但它们的结合并没有导致氧释放的恢复。相比之下，用缺少 C 末端一个残基的突变蛋白进行重建，显示出与用全长 12 kDa 蛋白质进行重建相同的氧释放恢复。 (5) 这些结果表明，12 kDa 蛋白质 C 末端赖氨酸的两个残基构成了一个重要的结构域，可最大限度地减少析氧所需的氯和钙。此外，蛋白质的 N 末端至少有 5 个残基，具有满足氯化物需求的次要功能。
The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl(-) and Ca(2+) ions but significantly in the presence of Cl(-) ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl(-) and Ca(2+) ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.
DOI：
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发表时间：
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期刊：
影响因子：
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作者：
通讯作者：
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影响因子：
2.9
作者：
Enami, I;Kikuchi, S;Shen, JR
通讯作者：
Shen, JR