Topology and sequence in the folding of a TIM barrel protein:: Global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (βα)8 barrel of unknown function from B-subtilis
Topology and sequence in the folding of a TIM barrel protein:: Global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (βα)8 barrel of unknown function from B-subtilis
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DOI：
10.1016/j.jmb.2007.06.018
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发表时间：
2007-09-07
影响因子：
5.6
通讯作者：
Matthews, C. Robert
中科院分区：
文献类型：
--
作者：
Forsyth, William R.;Bilsel, Osman;Matthews, C. Robert
通过可逆尿素变性和圆二色谱、荧光和时间分辨荧光各向异性谱相结合的方法，评价了链拓扑结构和氨基酸序列对枯草杆菌IOLI基因编码的未知功能的(βα)(8)Tim桶蛋白折叠的相对贡献。IOLi的平衡反应，除了天然物种和未折叠物种外，还包括稳定的中间体，具有显著的二级结构和稳定性，以及天然和中间态的自缔合形式。全局动力学分析表明，在折叠早期，未折叠状态在路径外折叠中间体和路径上平衡中间体之间进行分配。与另外两个TIM桶蛋白，嗜热性Sulfolobus solfararicus的吲哚-3-甘油磷酸合成酶(SIGPS)和大肠杆菌色氨酸合成酶的阿尔法亚单位(αTS)的折叠机制进行比较，发现了惊人的相似之处，这表明拓扑在折叠的早期和晚期都起着主导作用。序列特异性效应在松弛时间和相对稳定性的大小、αTS和sIGPS的额外单体折叠中间体的存在以及αTS的限速Pro异构化反应中都是明显的。(C)2007爱思唯尔有限公司。保留所有权利。
The relative contributions of chain topology and amino acid sequence in directing the folding of a (beta alpha)(8) TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene (IOLI) were assessed by reversible urea denaturation and a combination of circular dichroism, fluorescence and time-resolved fluorescence anisotropy spectroscopy. The equilibrium reaction for IOLI involves, in addition to the native and unfolded species, a stable intermediate with significant secondary structure and stability and self-associated forms of both the native and intermediate states. Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding. Comparisons with the folding mechanisms of two other TIM barrel proteins, indole-3-glycerol phosphate synthase from the thermophile Sulfolobus solfataricus (sIGPS) and the alpha subunit of Escherichia coli tryptophan synthase (alpha TS), reveal striking similarities that argue for a dominant role of the topology in both early and late events in folding. Sequence-specific effects are apparent in the magnitudes of the relaxation times and relative stabilities, in the presence of additional monomeric folding intermediates for alpha TS and sIGPS and in rate-limiting proline isomerization reactions for alpha TS. (c) 2007 Elsevier Ltd. All rights reserved.