Miranker A, Radford S E, Karplus M, Dobson C M
Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, Massachusetts 02138.
Nature. 1991 Feb 14;349(6310):633-6. doi: 10.1038/349633a0.
Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.
尽管人们对蛋白质折叠途径进行了大量推测，但直到最近才有关于该主题的实验数据。在完全折叠和未折叠状态之间的中间物种稳定的条件下对蛋白质进行研究，已经提供了重要信息，例如关于BPTI中的二硫键中间体、RNase A3的顺式/反式脯氨酸异构体以及α-乳白蛋白的熔融球状体状态。研究折叠途径的另一种方法涉及在重折叠研究中使用停流方法结合氢交换的NMR测量来检测和表征瞬态构象体。在细胞色素c、RNaseA和芽孢杆菌RNA酶折叠的早期阶段已经检测到中间结构的形成。对于α-乳白蛋白，通过NMR监测的氢交换动力学对于识别稳定熔融球状体状态下类似天然的结构特征至关重要。对于结构同源的蛋白质鸡蛋清溶菌酶，尚未观察到在平衡条件下稳定的类似部分折叠的蛋白质，尽管有证据表明在重折叠过程中形成了类似但瞬态的状态。在这里，我们描述了基于氢交换和重折叠过程之间竞争的NMR实验，这些实验不仅支持溶菌酶存在这样的瞬态物种，而且能够定义其结构特征。结果表明，溶菌酶的两个结构域是不同的折叠结构域，因为它们在折叠早期存在紧密的、可能类似天然的结构的程度上有显著差异。
