Whittaker Sara B-M, Spence Graham R, Günter Grossmann J, Radford Sheena E, Moore Geoffrey R
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, UK.
J Mol Biol. 2007 Feb 23;366(3):1001-15. doi: 10.1016/j.jmb.2006.11.012. Epub 2006 Nov 10.
Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native alpha-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.
先前的研究表明,Im7折叠过程中短暂出现的、处于折叠途径上的中间体包含四个天然α螺旋中的三个,它们围绕着一个由天然和非天然相互作用稳定的核心对接。为了更详细地确定该物种的结构和动力学性质,我们利用蛋白质工程技术在平衡状态下捕获中间体,并使用核磁共振光谱和小角X射线散射对所得蛋白质进行分析。创建了四个变体。在L53AI54A中,螺旋III内的两个疏水残基被截断,阻止螺旋III稳定地对接至正在形成的疏水核心上。在另外两个变体中,包含天然螺旋III的六个残基被三个(H3G3)或六个(H3G6)甘氨酸残基取代。在第四个变体YY中,将两个天然酪氨酸残基(Tyr55和Tyr56)重新引入H3G6中,以研究它们在确定中间体集合性质中的作用。所有四个变体均显示出可变的峰强度和宽峰宽,这与这些蛋白质在构象上具有动态性一致。化学位移分析表明,L53AI54A和YY在螺旋I和IV中含有类似天然的二级结构,而螺旋II部分形成,螺旋III不存在。L53AI54A缺乏核Overhauser效应(NOE)且NH交换迅速,结合对主链动力学的详细分析,表明该变体的疏水核心并非具有独特结构,而是在核磁共振时间尺度上波动。结果表明,尽管Im7的许多类似天然的二级结构存在于变体中,但其疏水核心仍然相对不稳定。H3G3/H3G6与L53AI54A/YY的比较表明,Tyr55和/或Tyr56与三螺旋核心相互作用,随着折叠的进行,导致该蛋白质区域中的其他残基与核心对接。在这方面,三螺旋束充当形成螺旋III和创建天然折叠的模板。