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首页> 外文期刊>Biochemistry >Structural and functional differentiation of three groups of tyrosine residues by acetylation of N-acetylimidazole in manganese stabilizing protein.
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Structural and functional differentiation of three groups of tyrosine residues by acetylation of N-acetylimidazole in manganese stabilizing protein.

机译：通过锰稳定蛋白中N-乙酰基咪唑的乙酰化作用，对三组酪氨酸残基进行结构和功能区分。

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摘要

To study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidazole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually approximately 5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried), and 1-2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that the 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of approximately 6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. Deacetylation of MSP with 6.4 or 8 acetylated Tyr residues with hydroxylamine restored most of the rebinding and oxygen-evolving activities. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.
机译：为了研究其对绿色植物锰稳定蛋白（MSP）组装到光系统II（PSII）中的贡献，使用N-乙酰基咪唑（NAI）对酪氨酸残基进行了特异性乙酰化。在可溶性MSP中，可以通过NAI乙酰化作用区分三组Tyr残基：大约5个（实际上约为5.2）Tyr残基可以很容易地被乙酰化（表面），当NAI浓度足够高时（表面上可以）1-2个Tyr残基可以被乙酰化。仅在变性剂尿素（深埋）的存在下才能乙酰化1-2个Tyr残基。 5.2 Tyr残基的乙酰化不影响MSP的重构或放氧活性，远紫外圆二色性（CD）分析表明，改变后的MSP保留了其大部分天然二级结构。这些结果表明5.2 Tyr残基不是MSP功能的绝对必需。但是，对1-2个表面掩埋的Tyr残基的进一步修饰（对于大约6.4 Tyr残基的总乙酰化）完全废除了MSP的重新结合和氧释放活性。最终，NAI无法接近至少一个酪氨酸残基，直到MSP被8 M尿素完全展开。用羟胺将6.4或8个乙酰化Tyr残基的MSP脱乙酰基恢复了大多数的重新结合和放氧活性。修饰6.4个Tyr残基后，观察到MSP的荧光光谱有明显的红移（在280或295 nm处激发），并且所有8个Tyr残基都被修饰后，发现进一步的红移，表明天然二级结构的损失很大。远紫外线CD显示，当修饰6.4个Tyr残基时，MSP大部分展开，而所有8个Tyr残基均被修饰完全展开。荧光和远紫外CD研究表明，NAI修饰后MSP重新结合到PSII膜上的损失与MSP的构象变化密切相关。总之，这些结果表明，不同的酪氨酸残基对MSP结合和组装成PSII的贡献不同。

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