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

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 formatio...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-11, Vol.275 (45), p.34968-34975
Main Authors: Samuel, Dharmaraj, Kumar, Thallampuranam Krishnaswamy Suresh, Srimathi, Thiagarajan, Hsieh, Hui-chu, Yu, Chin
Format: Article
Language:English
Subjects:
Anilino Naphthalenesulfonates - pharmacology
Chromatography
Densitometry
Deuterium - metabolism
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Fibroblast Growth Factor 1
Fibroblast Growth Factor 2 - chemistry
Fluorescent Dyes - pharmacology
Guanidine - pharmacology
Heparin - metabolism
Humans
Hydrogen - metabolism
Kinetics
Magnetic Resonance Spectroscopy
Models, Molecular
Protein Binding
Protein Conformation
Protein Folding
Protein Structure, Secondary
Spectrometry, Fluorescence
Time Factors
Urea - pharmacology
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M005147200