Ibarra-Molero B, Sanchez-Ruiz J M
Departamento de Quimica Fisica (Facultad de Ciencias) e Instituto de Biotecnologia, 18071-Granada, Spain.
Biochemistry. 1997 Aug 5;36(31):9616-24. doi: 10.1021/bi9703305.
Protein folding intermediates that are sometimes populated at equilibrium under mild denaturing conditions have attracted much attention as plausible models for the kinetic intermediates transiently populated in the refolding kinetic pathways. Hen egg-white lysozyme is often considered as a typical example of close adherence to the equilibrium, two-state unfolding mechanism. However, recent small-angle X-ray scattering studies suggest that an equilibrium intermediate state is significantly populated in the urea-induced unfolding of this protein at moderately acidic pH. In this work, we analyze the urea-induced unfolding of hen egg-white lysozyme on the basis of steady-state fluorescence measurements, characterization of the folding-unfolding kinetics, double-jump unfolding assays for the amount of native protein, and double-jump refolding assays for the amount of unfolded protein. Our results do not provide support for the presence of an intermediate state and, in particular, disfavor that the following two types of intermediates be significantly populated at equilibrium: (1) intermediates showing a substantial quenching of the tryptophan fluorescence (such as that observed in the transient intermediates found in the refolding kinetic pathway under strongly native conditions) and (2) associating intermediates. Also, the deconvolution of the radius of gyration unfolding profile by using the values for the amount of native state derived from our double-jump unfolding assays is consistent with a two-state unfolding equilibrium and suggests, furthermore, that, in this case, large alterations in the average structure of the unfolded ensemble do not take place in response to changes in urea concentration. This work points up possible pitfalls in the experimental detection of equilibrium folding intermediates and suggests procedures to circumvent them.
在温和变性条件下有时会在平衡状态下出现的蛋白质折叠中间体，作为在重折叠动力学途径中瞬时出现的动力学中间体的合理模型，已引起了广泛关注。鸡蛋清溶菌酶通常被认为是紧密遵循平衡的两态展开机制的典型例子。然而，最近的小角X射线散射研究表明，在中等酸性pH值下，该蛋白质在尿素诱导的展开过程中，平衡中间体状态大量出现。在这项工作中，我们基于稳态荧光测量、折叠-展开动力学的表征、天然蛋白质含量的双跳展开分析以及未折叠蛋白质含量的双跳重折叠分析，对尿素诱导的鸡蛋清溶菌酶展开进行了分析。我们的结果不支持中间体状态的存在，特别是不支持以下两种类型的中间体在平衡状态下大量出现：（1）色氨酸荧光有显著淬灭的中间体（如在强天然条件下重折叠动力学途径中发现的瞬时中间体中观察到的那样）和（2）缔合中间体。此外，通过使用我们的双跳展开分析得出的天然状态含量值对回转半径展开曲线进行去卷积，与两态展开平衡一致，并且进一步表明，在这种情况下，未折叠集合体的平均结构不会因尿素浓度的变化而发生大的改变。这项工作指出了在平衡折叠中间体的实验检测中可能存在的陷阱，并提出了规避这些陷阱的方法。
