Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein.
Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein.
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具体结构出现在色氨酸合酶（一种 TIM 桶状蛋白）α 亚基的亚毫秒折叠中间体的 N 末端。
DOI：
10.1016/j.jmb.2005.06.006
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Matthews,CRobert
中科院分区：
文献类型：
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作者：
Wu,Ying;Vadrevu,Ramakrishna;Yang,Xiaoyan;Matthews,CRobert
在许多球状蛋白质中观察到的亚毫秒折叠反应产物的相互竞争的观点被归因于离散的、部分折叠的状态的形成或自然有利条件下未折叠的链的随机断裂。为了验证这些对(βα)8，TIM桶基序中停流猝发相反应的解释的有效性，在大肠杆菌色氨酸合成酶(αTS)的α亚基的5个不同的亮氨酸或异亮氨酸残基上进行了一系列丙氨酸替换。这种蛋白质被认为在亚毫秒的时间范围内折叠成一个具有显著稳定性和∼50%的天然构象的远紫外圆二色(CD)信号的旁路中间体。α1、β2或β3中三个异亮氨酸或亮氨酸残基中的任何一个上的单个丙氨酸替换完全消除了非途径物种。这些变种在5ms内访问一种中间体，其性质与野生型αTS中中等尿素浓度下高度聚集的途径上平衡中间体的性质非常相似。相比之下，β4或β6中亮氨酸残基的丙氨酸替代会破坏稳定性，但保留了非途径的突变期物种。当考虑到互补的热力学和动力学数据时，这种突变分析表明，αTS的CD信号的亚毫秒出现反映了在不同热力学状态下二级结构的获得，而不是未折叠链的随机崩溃。连续的α1/β2/β3结构域以及C末端β4和β6链的替换的对比结果表明，爆发期物种具有异质性结构。α1/β2/β3结构域似乎紧密堆积，C末端表现为熔融球状结构，其折叠与α1/β2/β3结构域紧密耦合。
Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (βα)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (αTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and ∼50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either α1, β2 or β3 completely eliminate the off-pathway species. These variants, within 5ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type αTS. By contrast, alanine replacements for leucine residues in either β4 or β6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for αTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous α1/β2/β3 domain and the C-terminal β4 and β6 strands imply a heterogeneous structure for the burst-phase species. The α1/β2/β3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the α1/β2/β3 domain.