Cold denaturation of ubiquitin at high pressure

Cold‐induced conformational transition of ubiquitin was studied at pH 4.5 under a constant pressure of 2 kbar using variable pressure one‐dimensional 1 H and two‐dimensional 15 N/ 1 H 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 °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 °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 °C, whereas the IR spectrum shows a substantial decrease of the β‐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 1 H NMR data is fitted well with a single Δ C p value of 2.43 ± 0.13 (kJ/K mol) for the entire temperature range between − 21 and 90 °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 Δ C p value determined at 2 kbar is substantially smaller than the Δ C p determined at 1 bar (3.8–5.8 (kJ/K mol)), 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. Copyright © 2006 John Wiley & Sons, Ltd.