High pressure NMR reveals that apomyoglobin is an equilibrium mixture from the native to the unfolded.
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.
Kitahara
Ryo
R
Department of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
Yamada
Hiroaki
H
Akasaka
Kazuyuki
K
Wright
Peter E
PE
D001059:Apoproteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin