Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.
Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.
复制标题
牛催产素-神经素前体的表达、折叠和热力学特性：与分子间催产素-神经素复合物的关系。
DOI：
10.1021/bi9912950
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发表时间：
1999
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;Lu,M;Peyton,D;Breslow,E
早期对成熟催产素和神经物理蛋白之间分子间相互作用的热力学研究，以及这些相互作用对神经物理蛋白折叠的影响，提出了关于催产素和神经物理蛋白在其共同前体中的分子内相互作用的问题。为了解决这个问题，富含二硫键的催产素相关牛神经物理蛋白前体在大肠杆菌中表达，并在体外折叠产生毫克量的纯化蛋白；有证据表明，半胱氨酸残基的损害导致产量的显著阻碍。研究发现，在催产素存在的情况下，与降低的成熟神经物理素重折叠相关的低效在前体中没有得到缓解。与此一致的是，pH对前体光谱性质的影响以及对前体和成熟神经物理蛋白对胍变性的相对稳定性的影响表明，前驱体中催产素和神经物理蛋白之间的非共价分子内键仅比分子间复合体中的相应键具有较小的热力学优势。激素和蛋白质之间的主要相互作用的丧失，以及前体相对于成熟的未连接蛋白的稳定性的增强，随着pH的升高而可逆地发生，中点在pH 10。这些结果与核磁共振研究前体和分子间复合体之间的结构差异的证据相关联，这些差异在pH 10转变之后仍然存在，表明激素在前体中的共价结合需要其神经物理蛋白片段的构象变化，并导致系统的性质不同于连接或未连接的成熟蛋白的那些。
Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed inEscherichia coliand folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.