INTERMEDIATES IN THE REFOLDING OF REDUCED RIBONUCLEASE-A
INTERMEDIATES IN THE REFOLDING OF REDUCED RIBONUCLEASE-A
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DOI：
10.1016/0022-2836(79)90504-7
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发表时间：
1979-01-01
影响因子：
5.6
通讯作者：
CREIGHTON, TE
中科院分区：
文献类型：
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作者：
CREIGHTON, TE
在天然[牛胰] RNase 解折叠和还原蛋白重折叠过程中积累的具有 1、2、3 或 4 个二硫键的中间体通过用碘乙酸盐快速烷基化来捕获，并通过离子交换色谱法分离。它们在不同程度上被表征为酶活性、通过聚丙烯酰胺凝胶的电泳迁移率、半胱氨酸残基之间的二硫键、紫外吸收和荧光光谱所示的6个酪氨酸残基的环境、与针对捕获的未折叠还原蛋白或天然折叠蛋白的抗体的相互作用，以及尿素对任何稳定构象的破坏，从而产生分子形状的变化。正确重折叠的 RNase 与原始天然蛋白质无法区分，但实际上所有直接由还原蛋白质形成的具有最多 4 个二硫键的中间体均具有酶活性，并且根据这些标准未折叠。展开天然 RNase 是向完全还原蛋白质的全有或全无转变，没有二硫键中间体的积累。重折叠的中间体通过折叠转变中的最高能垒与完全折叠状态分离；它们可以被认为是未折叠蛋白质的可快速相互转化、相对不稳定的微观状态。最终构象的测量元素不是在第 3 个二硫键形成过程中获得的，而是与第 4 个天然二硫键形成同时出现。这些使用 RNase 的观察结果在质量上与之前使用胰腺胰蛋白酶抑制剂进行的更详细的观察结果相似，并提出了蛋白质解折叠和重折叠动力学过程的可能的一般模式。
The intermediates with 1, 2, 3 or 4 disulfide bonds which accumulate during unfolding of native [bovine pancreatic] RNase and refolding of the reduced protein were trapped by rapid alkylation with iodoacetate and separated by ion-exchange chromatography. They were characterized to varying extents by their enzymic activity, electrophoretic mobility through polyacrylamide gels, disulfide bonds between cysteine residues, the environments of the 6 tyrosine residues as indicated by UV absorption and fluorescence spectra, interaction with antibodies directed against either the trapped unfolded reduced protein or the native folded protein, and for the disruption by urea of any stable conformation producing a change in molecular shape. Correctly refolded RNase was indistinguishable from the original native protein, but virtually all the intermediates with up to 4 disulfide bonds formed directly from the reduced protein were enzymically inactive and unfolded by these criteria. Unfolding the native RNase was an all-or-none transition to the fully reduced protein, with no accumulation of disulfide intermediates. The intermediates in refolding are separated from the fully folded state by the highest energy barrier in the folding transition; they may be considered rapidly interconvertable, relatively unstable microstates of the unfolded protein. The measured elements of the final conformation are not acquired during formation of the 1st 3 disulfide bonds, but appear simultaneously with formation of the 4th native disulfide bond. These observations with RNase are qualitatively similar to those made previously in greater detail with pancreatic trypsin inhibitor and suggest a possible general pattern for the kinetic process of protein unfolding and refolding.