Eubanks S, Nguyen T L, Peyton D, Breslow E
Department of Biochemistry, The Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.
Biochemistry. 2000 Jul 11;39(27):8085-94. doi: 10.1021/bi0001527.
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 in Escherichia 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.
牛神经垂体素通常被视为神经垂体素行为的范例，其在二硫键配对的折叠状态下是亚稳态的，需要配体稳定化才能从还原状态有效折叠。对无配体猪神经垂体素（与催产素相关的类别）的研究表明，在中性pH条件下，其二聚化常数比相应的牛蛋白大90多倍，并且表明二聚化常数的增加伴随着稳定性的增加，足以使还原蛋白在没有配体肽的情况下有效折叠。通过对牛蛋白进行位点特异性诱变并在大肠杆菌中表达，发现牛蛋白和猪蛋白之间的功能差异仅归因于猪蛋白中的两个亚基界面突变，即第80位的组氨酸突变为精氨酸以及第81位的谷氨酸突变为苯丙氨酸。单独将His-80突变为Arg对二聚化的影响相对较小，而突变为Glu或Asp则在无配体状态下显著降低二聚化，尽管二聚化与结合之间的正向联系明显保留。对不同突变体的肽结合常数的比较还表明，His-80的取代导致结合亲和力和特异性的改变，这与对二聚化的影响无关。结果证明了亚基界面的羧基结构域片段在调节神经垂体素特性方面的重要性，并表明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有特定贡献。还指出了通过改变界面产生的自折叠和单体突变体对未来研究的潜在用途。
