Characterization of the structure and dynamics of a near-native equilibrium intermediate in the unfolding pathway of an all beta-barrel protein.
The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.
Srimathi
Thiagarajan
T
Department of Chemistry, National Tsing Hua University, Hsinchu 300 43, Taiwan.
Kumar
Thallampuranam Krishnaswamy S
TK
Chi
Ya-hui
YH
Chiu
Ing-Ming
IM
Yu
Chin
C
D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007554:Isotopes; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
D007554:Isotopes; D008024:Ligands; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D014357:Trypsin; D019791:Guanidine