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首页> 外文期刊>Journal of Molecular Biology >An obligatory intermediate controls the folding of the alpha-subunit of tryptophan synthase, a TIM barrel protein
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An obligatory intermediate controls the folding of the alpha-subunit of tryptophan synthase, a TIM barrel protein

机译：强制性中间体控制色氨酸合酶（一种TIM桶蛋白）的α亚基的折叠

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摘要

The proposed kinetic folding mechanism of the alpha-subunit of tryptophan Western synthase (alpha TS), a TIM barrel protein, displays multiple unfolded and intermediate forms which fold through four parallel pathways to reach the native state. To obtain insight into the secondary structure that stabilizes a set of late, highly populated kinetic intermediates, the refolding of urea-denatured alpha TS. from Escherichia coli was monitored by pulse-quench hydrogen exchange mass spectrometry. Following dilution from 8 M urea, the protein was pulse-labeled with deuterium, quenched with acid and mass analyzed by electrospray ionization mass spectrometry (ESI-MS). Hydrogen bonds that form prior to the pulse of deuterium offer protection against exchange and, therefore, retain protons at the relevant amide bonds. Consistent with the proposed refolding model, an intermediate builds up rapidly and decays slowly over the first 100 seconds of folding. ESI-MS analysis of the peptic fragments derived from alpha TS mass-labeled and quenched after two seconds of refolding indicates that the pattern of protection of the backbone amide hydrogens in this transient intermediate is very similar to that observed previously for the equilibrium intermediate of alpha TS highly populated at 3 M urea. The protection observed in a contiguous set of P-strands and a-helices in the N terminus implies a significant role for this sub-domain in directing the folding of this TIM barrel protein. (c) 2005 Elsevier Ltd. All rights reserved.
机译：色氨酸Western合酶（alpha TS）的TIM桶蛋白的alpha亚基的拟议动力学折叠机制显示出多种未折叠和中间形式，它们通过四个平行途径折叠而达到天然状态。为了深入了解可稳定一组后期高密度动力学中间体的二级结构，尿素变性的αTS的重折叠。通过脉冲猝灭氢交换质谱法监测来自大肠杆菌的大肠杆菌。从8 M尿素稀释后，蛋白质用氘进行脉冲标记，用酸淬灭，并通过电喷雾电离质谱（ESI-MS）进行质量分析。在氘脉冲之前形成的氢键可防止交换，因此可将质子保留在相应的酰胺键上。与建议的重折叠模型一致，中间体在折叠的前100秒内迅速聚集并缓慢衰减。 ESI-MS分析衍生自α-TS的消化片段经质量标记并在重新折叠两秒后淬灭，表明该过渡中间体中主链酰胺氢的保护模式与先前观察到的α平衡中间体非常相似TS的尿素浓度为3M。在N末端的一组连续P链和a螺旋中观察到的保护作用表明，该亚域在指导TIM桶蛋白折叠中起着重要作用。 （c）2005 Elsevier Ltd.保留所有权利。

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