Rozema D, Gellman S H
Department of Chemistry, University of Wisconsin, Madison 53706, USA.
Biochemistry. 1996 Dec 10;35(49):15760-71. doi: 10.1021/bi961638j.
Conditions that promote renaturation of an unfolded protein also promote protein aggregation, in many cases, because these competing intramolecular and intermolecular processes are driven by similar networks of noncovalent interactions. The GroEL/GroES system and related biological chaperones facilitate the renaturation of substrate proteins by minimizing the aggregation pathway. We have devised a two-step method in which small molecules, "artificial chaperones," facilitate protein refolding from a chemically denatured state. In the first step, the protein is captured by a detergent as guanidinium chloride is diluted to a non-denaturing concentration; formation of a protein-detergent complex prevents both protein aggregation and proper refolding. In the second step, a cyclodextrin strips detergent from the protein, allowing the protein to refold. Here we describe the first application of this method to a protein that must form disulfides in the native state. Lysozyme (hen egg white) can be refolded from the Gdm-denatured, DTT-reduced state in good yields at final protein concentrations as high as 1 mg/mL with the artificial chaperone method. Several mechanistic aspects of artificial chaperone-assisted refolding have been probed, and a detailed mechanism for the kinetically controlled stripping step is proposed.
在许多情况下，促进未折叠蛋白质复性的条件也会促进蛋白质聚集，因为这些相互竞争的分子内和分子间过程是由相似的非共价相互作用网络驱动的。GroEL/GroES系统及相关的生物伴侣蛋白通过使聚集途径最小化来促进底物蛋白的复性。我们设计了一种两步法，其中小分子“人工伴侣蛋白”促进化学变性状态下的蛋白质重折叠。第一步，随着氯化胍被稀释到非变性浓度，蛋白质被去污剂捕获；蛋白质-去污剂复合物的形成既能防止蛋白质聚集，也能防止正确重折叠。第二步，环糊精从蛋白质上去除去污剂，使蛋白质能够重折叠。在此，我们描述了该方法首次应用于在天然状态下必须形成二硫键的蛋白质。溶菌酶（鸡蛋清）采用人工伴侣蛋白方法，在最终蛋白质浓度高达1mg/mL时，能以较高产率从盐酸胍变性、二硫苏糖醇还原的状态重折叠。我们已经探究了人工伴侣蛋白辅助重折叠的几个机制方面，并提出了动力学控制的去除步骤的详细机制。
