Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants.
Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants.
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山羊 α-乳清蛋白及其 N 末端变体的连续四态折叠/展开。
DOI：
10.1002/prot.24109
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Kuwajima K.
中科院分区：
文献类型：
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作者：
Tomoyori K;Nakamura T;Makabe K;Maki K;Saeki K;Kuwajima K.
采用圆二色谱法研究了α-乳清蛋白及其两种N-末端变体的折叠/去折叠平衡和动力学。这两种变体是野生型重组体和在大肠杆菌中表达的Glu 1缺失（E1 M）变体。在重组α-乳清蛋白的N末端存在额外的甲硫氨酸使蛋白质不稳定2 kcal/mol，而在Glu 1被Met 1取代的E1 M变体中恢复了稳定性。由盐酸胍的停流浓度跳跃诱导的蛋白质的动力学折叠/解折叠反应表明在重折叠中存在爆发相，并且在折叠和解折叠分支中给出具有显著曲率的人字形图。根据爆发相中间体的累积来解释折叠肢曲率。然而，在解折叠动力学中未观察到用于解释解折叠-肢体曲率的爆发相。因此，我们假设了一个连续的四态机制，其中来自爆发相中间体的折叠发生在被高能中间体分开的两个过渡态上。我们估计了爆发相中间体和两个过渡态的自由能的变化，这是由N末端变化和稳定钙离子的存在引起的。由此获得的N末端和Ca 2+结合位点的Φ值在折叠过程中连续增加，证明了顺序机制的有效性。E1 M变体的稳定性和折叠行为与真实蛋白质基本相同，使我们能够在未来的研究中使用该变体作为假野生型α-乳清蛋白。Proteins 2012; © 2012 Wiley Periodicals，Inc.
Equilibria and kinetics of folding/unfolding of α‐lactalbumin and its two N‐terminal variants were studied by circular dichroism spectroscopy. The two variants were wild‐type recombinant and Glu1‐deletion (E1M) variants expressed inEscherichia coli. The presence of an extra methionine at the N terminus in recombinant α‐lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped‐flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst‐phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding‐limb curvature was interpreted in terms of accumulation of the burst‐phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding‐limb curvature. We thus assumed a sequential four‐state mechanism, in which the folding from the burst‐phase intermediate takes placeviatwo transition states separated by a high‐energy intermediate. We estimated changes in the free energies of the burst‐phase intermediate and two transition states, caused by the N‐terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca2+‐binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo‐wild‐type α‐lactalbumin in future studies. Proteins 2012; © 2012 Wiley Periodicals, Inc.