The dynamic equilibrium unfolding pathway of human tumor necrosis factor-α (TNF-α) 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-α 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-α unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-α, trimeric and all β-sheet protein, could not be viewed from the simple two state model (N→U). Copyright (C) 1999 Elsevier Science B.V.
|Number of pages||10|
|Journal||Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology|
|Publication status||Published - 1999 Jan 11|
Bibliographical noteFunding Information:
This research was supported by a grant from the G7 Project and the National Creative Research Initiatives (D. Kim) of the Ministry of Science and Technology, and Y.-R. Kim thanks for the partial support from CRM-KOSEF grant (98K3-0302-01-01-3), Republic of Korea.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Structural Biology