Gloss L M, Simler B R, Matthews C R
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4460, USA.
J Mol Biol. 2001 Oct 5;312(5):1121-34. doi: 10.1006/jmbi.2001.4974.
The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts.
二聚体大肠杆菌色氨酸阻遏物（TR）的折叠机制是一个动力学复杂的过程，涉及三个不同的发展阶段。在形成部分折叠的单体物种集合后，在5毫秒内，向天然二聚体的折叠由三个动力学阶段控制。每个阶段的限速步骤要么是非脯氨酸异构化反应，要么是二聚化反应，这取决于最终变性剂的浓度。已经采用了两种方法，通过三个平行通道来测试先前提出的TR折叠机制：（1）去折叠双跳实验表明，所有三个折叠通道都直接通向天然二聚体；（2）通过添加盐对折叠最后一步的过渡态和天然二聚体进行差异稳定化，表明所有三个通道都涉及二聚体物种的异构化。提出了一个色氨酸阻遏物折叠的改进模型，其中所有三个通道都涉及部分折叠单体之间的快速二聚化反应，随后二聚体中间体异构化以产生天然二聚体。部分折叠单体的集合可以在低pH下达到平衡时捕获；pH 2.5下的一维质子核磁共振光谱表明，单体以两种不同的、缓慢相互转化的构象存在。这些数据为TR的三通道折叠机制提供了一个潜在的结构解释：两种不同单体形式的随机缔合，它们通过核心二聚化结构域和DNA结合螺旋-转角-螺旋结构域的不同堆积模式来区分。这些堆积模式中的一种，也许两种，都包含非天然接触。
