Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride.
The dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha (TNF-alpha) during denaturation at different guanidine hydrochloride (GdnHCl) concentrations (0-4.2 M) was investigated by steady-state fluorescence spectroscopy, potassium iodide (KI) fluorescence quenching, far-UV circular dichroism (CD), picosecond time-resolved fluorescence lifetime, and anisotropy decay measurements. We utilized the intrinsic fluorescence of Trp-28 and Trp-114 to characterize the conformational changes involved in the equilibrium unfolding pathway. The detailed unfolding pathway under equilibrium conditions was discussed with respect to motional dynamics and partially folded structures. At 0-0.9 M [GdnHCl], the rotational correlation times of 22-25 ns were obtained from fluorescence anisotropy decay measurements and assigned to those of trimeric states by hydrodynamic calculation. In this range, the solvent accessibility of Trp residues increased with increasing [GdnHCl], suggesting the slight expansion of the trimeric structure. At 1.2-2.1 M [GdnHCl], the enhanced solvent accessibility and the rotational degree of freedom of Trp residues were observed, implying the loosening of the internal structure. In this [GdnHCl] region, TNF-alpha was thought to be in soluble aggregates having distinct conformational characteristics from a native (N) or fully unfolded state (U). At 4.2 M [GdnHCl], TNF-alpha unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-alpha, trimeric and all beta-sheet protein, could not be viewed from the simple two state model (N-->U).
Kim
Y R
YR
Department of Chemistry, Yonsei University, Seoul, South Korea. yrkim@alchemy.yonsei.ac.kr
Hahn
J S
JS
Hong
H
H
Jeong
W
W
Song
N W
NW
Shin
H C
HC
Kim
D
D
D016880:Anisotropy; D002021:Buffers; D019791:Guanidine; D006801:Humans; D011193:Potassium Iodide; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*