High pressure NMR reveals that apomyoglobin is an equilibrium mixture from the native to the unfolded |
| |
Authors: | Kitahara Ryo Yamada Hiroaki Akasaka Kazuyuki Wright Peter E |
| |
Institution: | Department of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. |
| |
Abstract: | Pressure-induced reversible conformational changes of sperm whale apomyoglobin have been studied between 30 bar and 3000 bar on individual residue basis by utilizing 1H/15N hetero nuclear single-quantum coherence two-dimensional NMR spectroscopy at pH 6.0 and 35 degrees C. Apomyoglobin showed a series of pressure-dependent NMR spectra as a function of pressure, assignable to the native (N), intermediates (I), molten globule (MG) and unfolded (U) conformers. At 30 bar, the native fold (N) shows disorder only in the F helix. Between 500 bar and 1200 bar, a series of locally disordered conformers I are produced, in which local disorder occurs in the C helix, the CD loop, the G helix and part of the H helix. At 2000 bar, most cross-peaks exhibit severe line-broadening, suggesting the formation of a molten globule, but at 3000 bar all the cross-peaks reappear, showing that the molten globule turns into a well-hydrated, mobile unfolded conformation U. Since all the spectral changes were reversible with pressure, apomyoglobin is considered to exist as an equilibrium mixture of the N, I, MG and U conformers at all pressures. MG is situated at 2.4+/-(0.1) kcal/mol above N at 1 bar and the unfolding transition from the combined N-I state to MG is accompanied by a loss of partial molar volume by 75+/-(3) ml/mol. On the basis of these observations, we postulate a theorem that the partial molar volume of a protein decreases in parallel with the loss of its conformational order. |
| |
Keywords: | HSQC hetero nuclear single-quantum coherence ΔV partial molar volume ΔG° Gibbs energy difference at 1 bar N native conformer I locally unfolded intermediates MG molten globule U unfolded conformer |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|