Lu H, Buck M, Radford S E, Dobson C M
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, UK.
J Mol Biol. 1997 Jan 17;265(2):112-7. doi: 10.1006/jmbi.1996.0715.
The refolding of a partially structured state of hen lysozyme formed in 60% (v/v) 2,2,2-trifluoroethanol (TFE) has been studied using hydrogen exchange pulse labelling monitored by 2D 1H NMR, and by stopped flow fluorescence and CD measurements. The results are compared with similar studies of the refolding of the protein denatured in 6 M guanidine hydrochloride (GuHCl). Two conclusions have emerged from these studies. First, provided that the refolding conditions are identical, the two denatured states fold with very similar kinetics, despite the fact the extensive secondary structure is present in the TFE-denatured state but not in the protein denatured in 6 M GuHCl. This arises because of the rapid equilibration of structure in the species formed in the initial stage of folding. Second, whilst addition of GuHCl to the refolding buffer decreases the rate of folding, low concentrations of TFE increase the rate of folding. The result is consistent with slow steps in the refolding of lysozyme being associated primarily with the reorganisation of hydrophobic interactions rather than of hydrogen bonded structure.
利用二维¹H NMR监测的氢交换脉冲标记法、停流荧光法和圆二色性测量法，研究了在60%（v/v）2,2,2-三氟乙醇（TFE）中形成的部分结构化状态的鸡溶菌酶的重折叠过程。将结果与在6 M盐酸胍（GuHCl）中变性的该蛋白质重折叠的类似研究进行了比较。这些研究得出了两个结论。首先，只要重折叠条件相同，尽管TFE变性状态中存在广泛的二级结构，而在6 M GuHCl中变性的蛋白质中不存在，但这两种变性状态以非常相似的动力学进行折叠。这是由于在折叠初始阶段形成的物种中结构的快速平衡所致。其次，虽然向重折叠缓冲液中添加GuHCl会降低折叠速率，但低浓度的TFE会提高折叠速率。该结果与溶菌酶重折叠过程中的慢步骤主要与疏水相互作用的重新组织而非氢键结构的重新组织相关一致。
