Intramolecular Cross-linking of the Extrinsic 33-kDa Protein Leads to Loss of Oxygen Evolution but Not Its Ability of Binding to Photosystem II and Stabilization of the Manganese Cluster*
Intramolecular Cross-linking of the Extrinsic 33-kDa Protein Leads to Loss of Oxygen Evolution but Not Its Ability of Binding to Photosystem II and Stabilization of the Manganese Cluster*
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DOI：
10.1074/jbc.273.8.4629
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发表时间：
1998-02
期刊：
影响因子：
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通讯作者：
I. Enami;M. Kamo;H. Ohta;Seitaro Takahashi;Taro Miura;Miho Kusayanagi;Sizuko Tanabe;A. Kamei;A. Motoki;M. Hirano;T. Tomo;K. Satoh
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文献类型：
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作者：
I. Enami;M. Kamo;H. Ohta;Seitaro Takahashi;Taro Miura;Miho Kusayanagi;Sizuko Tanabe;A. Kamei;A. Motoki;M. Hirano;T. Tomo;K. Satoh
光系统II（PSII）的外源性33-kDa蛋白质通过零长度交联剂1-乙基-3-（3-二甲基氨基丙基）碳二亚胺分子内交联。所得到的交联的33-kDa蛋白质反弹到尿素/NaCl洗涤的PSII膜，其稳定锰的结合有效地作为未处理的33-kDa蛋白质。与此相反，氧释放没有恢复的交联蛋白质的结合，这表明33 kDa蛋白质的结合和锰稳定的能力被保留，但其再活化能力是失去了分子内交联的蛋白质。从高温下CD光谱的测量，发现分子内交联的33-kDa蛋白质的二级结构在比未处理的33-kDa蛋白质高20 °C的温度下的热处理中稳定，表明33-kDa蛋白质的结构柔性通过分子内交联大大降低。刚性结构可能是负责的33-kDa蛋白的再活化能力的损失，这意味着，结合的33-kDa蛋白PSII是伴随着构象变化的再活化的氧释放必不可少的。肽图谱，N-末端测序，和质谱分析的蛋白酶消化产物的分子内交联的33-kDa蛋白质显示，交联发生在氨基之间的赖氨酸48和羧基的谷氨酸246，和羧基之间的谷氨酸10和氨基之间的赖氨酸14。因此，这些交联的氨基酸残基通过静电相互作用彼此紧密结合。
The extrinsic 33-kDa protein of photosystem II (PSII) was intramolecularly cross-linked by a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting cross-linked 33-kDa protein rebound to urea/NaCl-washed PSII membranes, which stabilized the binding of manganese as effectively as the untreated 33-kDa protein. In contrast, the oxygen evolution was not restored by binding of the cross-linked protein, indicating that the binding and manganese-stabilizing capabilities of the 33-kDa protein are retained but its reactivating ability is lost by intramolecular cross-linking of the protein. From measurements of CD spectra at high temperatures, the secondary structure of the intramolecularly cross-linked 33-kDa protein was found to be stabilized against heat treatment at temperatures 20 °C higher than that of the untreated 33-kDa protein, suggesting that structural flexibility of the 33-kDa protein was much decreased by the intramolecular cross-linking. The rigid structure is possibly responsible for the loss of the reactivating ability of the 33-kDa protein, which implies that binding of the 33-kDa protein to PSII is accompanied by a conformational change essential for the reactivation of oxygen evolution. Peptide mapping, N-terminal sequencing, and mass spectroscopic analysis of protease-digested products of the intramolecularly cross-linked 33-kDa protein revealed that cross-linkings occurred between the amino group of Lys48 and the carboxyl group of Glu246, and between the carboxyl group of Glu10and the amino group of Lys14. These cross-linked amino acid residues are thus closely associated with each other through electrostatic interactions.