Folding mechanism of the α-subunit of tryptophan synthase, an α/β barrel protein:: Global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels
Folding mechanism of the α-subunit of tryptophan synthase, an α/β barrel protein:: Global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels
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DOI：
10.1021/bi982365q
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发表时间：
1999-01-19
期刊：
影响因子：
2.9
通讯作者：
Matthews, CR
中科院分区：
文献类型：
--
作者：
Bilsel, O;Zitzewitz, JA;Matthews, CR
各种技术已被用来研究尿素诱导的动力学折叠机制的色氨酸合酶的a-亚基从大肠杆菌。这个29 kDa的α/β桶蛋白的一个独特的属性是存在两个稳定的平衡中间体，填充在约3和5 M尿素，重折叠过程显示多个动力学阶段，其寿命跨越亚毫秒到大于100秒的时间尺度;展开研究产生两个弛豫时间的顺序为10-100秒。为了了解稳定和瞬态中间体的种群和结构特性，将停流、手动混合和平衡圆二色性数据全局拟合到各种动力学模型。不同初始尿素浓度的复性和去折叠实验以及正向和反向双跳实验是模型判别的关键。最简单的动力学模型，这是与所有可用的数据一致，涉及四个缓慢相互转化的未折叠的形式，在5毫秒内崩溃的一个边缘稳定的中间体与显着的二级结构。这种早期中间体是一种非途径物质，必须展开以形成一组四种途径上的中间体，对应于3 M尿素平衡中间体。这些构象之间的再平衡作为速率限制步骤折叠的人口的大多数。在25摄氏度下，天然构象的一部分在不到1秒的时间内出现，这表明即使是大的蛋白质也可以快速穿过复杂的能量表面。
A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the a-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea, The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.