Conformational properties of the unfolded state of Im7 in nondenaturing conditions.
Conformational properties of the unfolded state of Im7 in nondenaturing conditions.
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DOI：
10.1016/j.jmb.2011.12.041
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发表时间：
2012-02-17
影响因子：
5.6
通讯作者：
Radford, Sheena E.
中科院分区：
文献类型：
--
作者：
Pashley, Clare L.;Morgan, Gareth J.;Kalverda, Arnout P.;Thompson, Gary S.;Kleanthous, Colin;Radford, Sheena E.
水溶液中未折叠的集合代表了蛋白质折叠的起点。这一物种的特征往往是困难的，因为原生状态通常是在平衡状态下占主导地位。先前的研究表明，四螺旋蛋白Im7（免疫蛋白7）通过一种通路上的中间体折叠。虽然过渡态和折叠中间体已经在原子细节上进行了表征，但在相同的环境条件下，对未折叠系综的了解仍然很少。在这里，我们在Im7序列中引入了不稳定的氨基酸取代，这样在没有变性剂的情况下，未折叠状态在平衡状态下变得主要填充。通过远紫外和近紫外CD，荧光，尿素滴定和异核磁共振实验，我们发现三个氨基酸取代（L18A-L19A-L37A）足以阻止Im7折叠，因此未折叠态主要分布在平衡态。通过测量化学位移、15N横向弛豫速率和沉降系数，我们发现L18A-L19A-L37A的未折叠态明显偏离了随机线圈的行为。具体来说，我们证明了这种未折叠的物种相对于尿素变性状态（Rh≥30 Å）是致密的（Rh = 25 Å），并且在与天然状态下的四个螺旋相对应的区域中含有局部疏水残基簇。尽管存在这些相互作用，但没有证据表明存在长期稳定的三级相互作用或持久的螺旋结构。结果显示，在天然状态下，多肽链最终形成螺旋I， II和IV的区域受到构象限制的未折叠的集合。在非变性条件下创造了Im7的未折叠变体。►这种变体是塌陷的，但缺乏固定的二级或三级结构。短暂的螺旋状和折叠使蛋白质开始折叠。
The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, 15N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (Rh = 25 Å) relative to the urea-denatured state (Rh ≥ 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state. ► An unfolded variant of Im7 has been created in nondenaturing conditions. ► The variant is collapsed but lacks fixed secondary or tertiary structure. ► Transient helicity and collapse prime the protein towards folding.
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影响因子：
5.6
作者：
Gorski, SA;Capaldi, AP;Radford, SE
通讯作者：
Radford, SE
影响因子：
5.6
作者：
ALEXANDRESCU, AT;SHORTLE, D
通讯作者：
SHORTLE, D
DOI：
10.1073/pnas.1101752108
发表时间：
2011-04-05
影响因子：
11.1
作者：
Banachewicz, Wiktor;Religa, Tomasz L.;Fersht, Alan R.
通讯作者：
Fersht, Alan R.
影响因子：
5.6
作者：
Hofmann, Hagen;Golbik, Ralph P.;Ulbrich-Hofmann, Renate
通讯作者：
Ulbrich-Hofmann, Renate
DOI：
10.1073/pnas.0609717104
发表时间：
2007-01-02
影响因子：
11.1
作者：
Huang, Fang;Sato, Satoshi;Fersht, Alan R.
通讯作者：
Fersht, Alan R.