Cold-induced conformational transition of ubiquitin was studied at pH 4.5 under a constant pressure of 2 kbar using variable pressure one-dimensional 1H and two-dimensional 15N/1H NMR spectroscopy as well as IR spectroscopy. Although a tendency for preferential stabilization of a peculiar locally disordered and partially hydrated conformer I, identical with that previously found with variable-pressure NMR at 0 degrees C, is recognized, the transition of the folded conformer N to the unfolded conformer U occurs largely cooperatively with decreasing temperature, reaching near completion at - 21 degrees C. NMR spectral features as well as the analysis of NMR relaxation parameters indicate that the polypeptide chain is almost fully unfolded, fairly well-hydrated and floppy at - 21 degrees C, whereas the IR spectrum shows a substantial decrease of the beta-sheet. The Gibbs energy change from the folded state (a mixture of N and I) to the unfolded state at 2 kbar obtained from the 1H NMR data is fitted well with a single DeltaCp value of 2.43 +/- 0.13 (kJ/K mol) for the entire temperature range between - 21 and 90 degrees C, covering both the cold denaturation and heat denaturation, showing that the two denatured states actually belong to a single thermodynamic phase of the protein. The DeltaCp value determined at 2 kbar is substantially smaller than the DeltaCp determined at 1 bar (3.8-5.8 (kJ/Kmol), which is consistent with the fact that the denaturation takes place from a mixture of N and I at 2 kbar rather than from pure N at 1 bar.

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