Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface.
Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface.
复制标题
通过神经素亚基界面的突变调节二聚化、结合、稳定性和折叠。
DOI：
10.1021/bi0001527
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Breslow,E
中科院分区：
文献类型：
--
作者：
Eubanks,S;Nguyen,TL;Peyton,D;Breslow,E
牛神经物理蛋白通常被用作神经物理蛋白行为的范例，它在二硫键对折叠状态下是亚稳定的，需要配体稳定才能从还原状态有效折叠。对未连接的猪神经物理蛋白(催产素相关类)的研究表明，在中性pH下，它的二聚化常数比相应的牛蛋白大90多倍，并表明二聚化常数的增加伴随着稳定性的增加，足以在没有配位肽的情况下有效折叠还原的蛋白质。利用牛蛋白的定点突变和在大肠杆菌中的表达，牛和猪蛋白之间的功能差异被证明完全归因于猪蛋白中的两个亚基界面突变，即第80位His到Arg和第81位Glu到Phe。单独突变His-80到Arg对二聚化的影响相对较小，而突变到Glu或Asp显著减少去连接状态下的二聚化，尽管明显保留了二聚化和结合之间的正向联系。对不同突变体的多肽结合常数的比较还表明，His-80的取代导致了结合亲和力和特异性的改变，而这些改变与二聚化的影响无关。结果证明了亚基界面的羧基结构域片段在调节神经物理性质中的重要性，并表明了该区域配体诱导的构象变化的能量学对结合的整体热力学的特定贡献。指出了通过改变界面产生的自折叠和单体突变的未来研究的潜在用途。
Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression inEscherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.