Mutagenesis of basic residues R151 and R161 in manganese-stabilizing protein of photosystem II causes inefficient binding of chloride to the oxygen-evolving complex.
Mutagenesis of basic residues R151 and R161 in manganese-stabilizing protein of photosystem II causes inefficient binding of chloride to the oxygen-evolving complex.
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DOI：
10.1021/bi0523759
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发表时间：
2006-02
期刊：
影响因子：
2.9
通讯作者：
H. Popelková;S. Betts;Nikos Lydakis-Symantiris;M. Im;Ellen E Swenson;C. Yocum
中科院分区：
文献类型：
--
作者：
H. Popelková;S. Betts;Nikos Lydakis-Symantiris;M. Im;Ellen E Swenson;C. Yocum
光系统II的锰稳定蛋白是一种本质上无序的多肽，含有高比例的带电和疏水性氨基酸残基。Arg 151和Arg 161在所有已知的MSP序列中是保守的。为了研究这些碱性残基在MSP结构和功能中的作用，产生了菠菜MSP的三种突变体R151 G、R151 D和R161 G。在这里，我们提出的证据表明，Arg 151或Arg 161的替代产生的蛋白质，具有较低的PSII结合亲和力，功能缺陷，即使约2摩尔的突变MSP/摩尔PSII可以反弹到MSP耗尽PSII膜。R161 G使O（2）释放活性恢复到对照的40%，而R151 G和R151 D仅恢复到对照的20%。光谱和生物化学技术未能检测到溶液结构的显著变化。更广泛的O（2）进化分析显示，Mn簇在用每个突变MSP重建的样品中是稳定的，并且所有三个Arg突变体具有与野生型蛋白相同的保留Ca（2+）的能力。然而，探索这些突变对Cl（-）保留的影响的活性测定显示，R151 G、R151 D和R161 G MSP在Cl（-）与OEC的结合方面存在缺陷。突变体的Cl（-）K（M）值比野生型蛋白质的值高约4倍（R161 G）或6倍（R151 G和R151 D）。本文报道的结果表明，锰稳定蛋白上的保守正电荷在蛋白质到PSII的适当功能组装中发挥作用，因此，在保留Cl（-）的O（2）-进化复合物。
Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Arg151 and Arg161 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced. Here, we present evidence that replacement of Arg151 or Arg161 yields proteins that have lower PSII binding affinity, and are functionally deficient even though about 2 mol of mutant MSP/mol PSII can be rebound to MSP depleted PSII membranes. R161G reconstitutes O(2) evolution activity to 40% of the control, while R151G and R151D reconstitute only 20% of the control activity. Spectroscopic and biochemical techniques fail to detect significant changes in solution structure. More extensive O(2) evolution assays revealed that the Mn cluster is stable in samples reconstituted with each mutated MSP, and that all three Arg mutants have the same ability to retain Ca(2+) as the wild-type protein. Activity assays exploring the effect of these mutations on retention of Cl(-), however, showed that the R151G, R151D, and R161G MSPs are defective in Cl(-) binding to the OEC. The mutants have Cl(-) K(M) values that are about four (R161G) or six times (R151G and R151D) higher than the value for the wild-type protein. The results reported here suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and, consequently, in retention of Cl(-) by the O(2)-evolving complex.