Absence of a stable intermediate on the folding pathway of protein A
Absence of a stable intermediate on the folding pathway of protein A
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DOI：
10.1002/pro.5560060709
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发表时间：
1997-07-01
期刊：
影响因子：
8
通讯作者：
Wright, PE
中科院分区：
文献类型：
--
作者：
Bai, YW;Karimi, A;Wright, PE
蛋白A的B结构域具有最简单的蛋白质拓扑结构之一，即三螺旋束。它的折叠已被研究作为一个模型的基本步骤，在折叠较大的蛋白质。早期的研究表明，折叠可能通过螺旋发夹中间体发生。平衡氢交换测量表明，C-末端螺旋发夹可能是一个潜在的折叠中间体。采用停流圆二色性和核磁共振氢氘交换脉冲标记进行动力学复性实验。整个分子的折叠基本上在现在淬灭装置的6 ms死时间内完成，表明中间体（如果形成的话）迅速进展到最终折叠状态。使用位置16处的异亮氨酸残基的定点诱变来产生含有色氨酸的变体蛋白（I16 W突变体）。由于预测的折叠状态下庞大色氨酸侧链的不利空间相互作用，预计在该突变体中以天然折叠形式为代价形成推定的折叠中间体是有利的。I16 W突变体在停流荧光实验的死时间内完全重新折叠。没有部分折叠的中间体可以检测到的动力学或平衡测量。肽片段的研究表明，蛋白A序列具有形成螺旋II/螺旋III发夹的内在倾向。然而，它的稳定性似乎是边际（1/2kT的顺序），它不能是一个强制性的中间体上定义的折叠途径。这些结果明确表明，蛋白质A B结构域折叠非常迅速的一个明显的两态机制，没有形成稳定的部分折叠的中间体。类似的机制也可能涉及较大蛋白质的亚结构域的快速折叠，以形成经常在折叠过程中积累的紧凑的熔融球中间体。
The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-now apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the I16W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The I16W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.