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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and ~(19)F NMR
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Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and ~(19)F NMR

机译：使用慢折叠突变体和〜（19）F NMR观察肠道脂肪酸结合蛋白折叠的顺序步骤

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摘要

The rat intestinal fatty acid binding protein (IFABP) primarily comprises two β-sheet structures surrounding a large internal cavity. The urea denatured WT protein folds within seconds after dilution to nondenaturing conditions. Replacing a glycine with valine in the turn between the last two β-strands (Gly121 Val) slows the folding process by more than three orders of magnitude. After incorporating 4-~(19)F-phenylalanine into the mutant protein, we were able to directly monitor the behavior of the eight phenylalanine rnside chains in real time during folding using ~(19)F NMR. Specifically, there is a nonnative-like collapse in regions involving three phenylalanine residues (Phe-62, Phe-68, and Phe-93) within milliseconds. At least two distinct NMR peaks were observed, suggesting conformational fluctuations in this region. Formation of this site is followed by formation of native structure of Phe-2 and Phe-17, then by Phe-47, and finally by the cooperative rearrangement of the intermediate structures to the final native structure. It is proposed that the Gly121 Val mutation slows the formation of a normal nucleating site, not only slowing overall folding, but also allowing intermediates in regions distant from the mutation to be experimentally observed. Because intermediates involved in protein folding are normally difficult to observe due to their marginal stability, the experimental approach used here may serve as a general method for determining the nature of both early and late steps in protein folding.
机译：大鼠肠道脂肪酸结合蛋白（IFABP）主要包括围绕大内腔的两个β-折叠结构。稀释至非变性条件后，尿素变性的WT蛋白在数秒内折叠。在最后两个β链（Gly121 Val）之间依次用缬氨酸取代甘氨酸可使折叠过程减慢三个数量级以上。将4-〜（19）F-苯丙氨酸掺入突变蛋白后，我们能够使用〜（19）F NMR直接实时监测折叠过程中八个苯丙氨酸侧链的行为。具体而言，在毫秒内涉及三个苯丙氨酸残基（Phe-62，Phe-68和Phe-93）的区域中会出现非自然样的塌陷。观察到至少两个不同的NMR峰，表明该区域的构象波动。该位点的形成之后是Phe-2和Phe-17的天然结构的形成，然后是Phe-47的形成，最后是中间结构向最终天然结构的协同重排。有人提出，Gly121 Val突变会减慢正常成核位点的形成，不仅减慢整体折叠，而且允许通过实验观察到远离突变的区域中的中间体。由于参与蛋白质折叠的中间体由于其边缘稳定性通常很难观察到，因此此处使用的实验方法可作为确定蛋白质折叠早期和晚期步骤性质的通用方法。

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