[[abstract]]The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all β-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy. The unfolding of hFGF-1 in GdnHCl is shown to involve the formation of a stable equilibrium intermediate. Size exclusion chromotagraphy using fast protein liquid chromatography shows that the intermediate accumulates maximally at 0.96 M GdnHCl. 1-Anilinonapthalene 8-sulfonate binding, one-dimensional 1H NMR, and limited proteolytic digestion experiments suggest that the intermediate has characteristics resembling a molten globule state. Chemical shift perturbation and hydrogen-deuterium exchange monitored by 1H-15N heteronuclear single quantum coherence spectra reveal that profound structural changes in the intermediate state (in 0.96 M GdnHCl) occur in the C-terminal, heparin binding region of the protein molecule. Additionally, results of the stopped flow fluorescence experiments suggest that the kinetic refolding of hFGF-1 proceeds through the accumulation of an intermediate at low concentrations of the denaturant. To our knowledge, the present study is the first report wherein an equilibrium intermediate is characterized in detail in an all β-barrel protein.
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机译:[[摘要]]使用多种生物物理技术(包括多维NMR光谱法)研究了盐酸胍(GdnHCl)诱导的全β-折叠蛋白(人酸性成纤维细胞生长因子(hFGF-1))的折叠。 hFGF-1在GdnHCl中的展开显示涉及稳定平衡中间体的形成。使用快速蛋白液相色谱的尺寸排阻色谱表明,中间体最大积累浓度为0.96 M GdnHCl。 1-Anilinonapthalene 8-磺酸盐结合,一维1 H NMR和有限的蛋白水解消化实验表明,该中间体具有类似于熔融球状状态的特征。通过1H-15N异核单量子相干光谱监测的化学位移扰动和氢-氘交换表明,中间态(在0.96 M GdnHCl中)的深刻结构变化发生在蛋白质分子的C端肝素结合区。此外,停止流动荧光实验的结果表明,hFGF-1的动力学重折叠通过低浓度变性剂中中间体的积累而进行。据我们所知,本研究是第一个报道,其中在所有β-桶蛋白中均详细描述了一种平衡中间体。
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