Identification and characterization of an equilibrium intermediate in the unfolding pathway of an all beta-barrel protein.
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 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 (1)H 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 (1)H-(15)N 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 beta-barrel protein.
Samuel
D
D
Department of Chemistry, National Tsing Hua University, Hsinchu, 30043 Taiwan.
Kumar
T K
TK
Srimathi
T
T
Hsieh
H
H
Yu
C
C
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D002845:Chromatography; D003720:Densitometry; D003903:Deuterium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D016220:Fibroblast Growth Factor 1; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; C027132:1-anilino-8-naphthalenesulfonate; D006859:Hydrogen; D014508:Urea; D006493:Heparin; D003903:Deuterium; D019791:Guanidine