Identification and Characterization of an Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

摘要: The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all beta-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety biophysical techniques including multidimensional NMR spectroscopy. hFGF-1 in GdnHCl shown to involve formation stable equilibrium intermediate. Size exclusion chromotagraphy fast protein liquid chromatography shows that intermediate accumulates maximally at 0.96 m GdnHCl. 1-Anilinonapthalene 8-sulfonate binding, one-dimensional (1)H NMR, and limited proteolytic digestion experiments suggest has characteristics resembling molten globule state. Chemical shift perturbation hydrogen-deuterium exchange monitored by (1)H-(15)N heteronuclear single quantum coherence spectra reveal profound structural changes state (in GdnHCl) occur C-terminal, heparin binding region molecule. Additionally, results stopped flow fluorescence kinetic refolding proceeds through accumulation low concentrations denaturant. To our knowledge, present study first report wherein characterized detail beta-barrel protein.