Hanson P E, Gellman S H
Department of Chemistry, University of Wisconsin, Madison 53706-1396, USA.
Fold Des. 1998;3(6):457-68. doi: 10.1016/S1359-0278(98)00063-7.
We have previously described a method for the refolding of chemically denatured proteins in which small molecules ('artificial chaperones', a detergent and cyclodextrin) assist renaturation. In a previous analysis of lysozyme refolding from the GdmCl-denatured, DTT-reduced state, we found that enzymatic activity is regained at indistinguishable rates for unassisted (absence of additives) and artificial-chaperone-assisted refolding. While unassisted and artificial-chaperone-assisted refolding rates could also be directly compared for GdmCl-denatured bovine carbonic anhydrase B (CAB), only cationic detergents could be used as assistants. We therefore set out to determine whether artificial chaperones could assist the refolding of urea-denatured CAB, whether the charge and structure of the detergent used affects refolding assistance, and, if so, whether the assistance is mechanistically similar to that observed for GdmCl-denatured CAB.
Our results indicate that CAB can be refolded from the urea-denatured state via the artificial chaperone process, using both anionic and cationic detergents. There is a distinctive product-determining step early in the artificial-chaperone-assisted refolding mechanism, but the rate-determining steps of the unassisted and artificial-chaperone-assisted processes are indistinguishable.
Because the rate-determining steps of unassisted and artificial-chaperone-assisted refolding are indistinguishable, we conclude that the rate-determining step of CAB refolding is unaffected by the use of artificial chaperones. Our observations also suggest that denatured CAB undergoes a slow partial folding in concentrated urea solution.
我们之前描述了一种化学变性蛋白质复性的方法，其中小分子（“人工伴侣”，一种去污剂和环糊精）协助复性。在之前对从盐酸胍变性、二硫苏糖醇还原状态复性的溶菌酶的分析中，我们发现对于无辅助（无添加剂）和人工伴侣辅助复性，酶活性以难以区分的速率恢复。虽然对于盐酸胍变性的牛碳酸酐酶B（CAB），也可以直接比较无辅助和人工伴侣辅助的复性速率，但只能使用阳离子去污剂作为辅助剂。因此，我们着手确定人工伴侣是否可以协助尿素变性的CAB复性，所用去污剂的电荷和结构是否会影响复性辅助，如果是，这种辅助在机制上是否与盐酸胍变性的CAB所观察到的相似。
我们的结果表明，使用阴离子和阳离子去污剂，CAB都可以通过人工伴侣过程从尿素变性状态复性。在人工伴侣辅助复性机制的早期有一个独特的产物决定步骤，但无辅助和人工伴侣辅助过程的速率决定步骤难以区分。
由于无辅助和人工伴侣辅助复性的速率决定步骤难以区分，我们得出结论，CAB复性的速率决定步骤不受人工伴侣使用的影响。我们的观察结果还表明，变性的CAB在浓尿素溶液中会经历缓慢的部分折叠。
