Structure of Pressure-Assisted Cold Denatured Lysozyme and Comparison with Lysozyme Folding Intermediates

Abstract

At high (> 3.5 kbar) pressures and low (< -10 degrees C) temperatures, hen egg-white lysozyme denatures readily and reversibly. Amide hydrogen exchange methods were used to investigate the structure of the pressure-assisted cold-denatured state of lysozyme. Protection factors were obtained for 52 backbone amide protons. The extent of protection of many of these protons is markedly different from that in lysozyme denatured by high temperature, high urea concentration, or chemical modification; specifically, the protection factors are higher and are strongly correlated with elements of secondary structure present in the native state. Furthermore, the pattern of protection factors is similar to that observed in lysozyme during refolding from highly denatured states, particularly during the early stages (< 3.5 ms) of refolding [Gladwin, S. T., & Evans, P. A. (1996) Folding Des. 1, 407]. Previous data on cold-denatured ribonuclease A were reevaluated and compared to known folding intermediates [Houry, W. A. & Scheraga, H. A. (1996) Biochemistry 35, 11734; Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197] to further test the supposition that the pressure-assisted cold-denatured states of proteins resemble the early folding stages.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

“…The P-factors determined in the present study for the cold-denatured state at -13ºC and 3.75 kbar qualitatively agree with those reported by Nash and Jonas (8), with high protection found at the cross-linked cysteine C64 and in the D-helix around C115, but the absolute values found in the current study were higher for most residues and in particular the P-factor determined for the crosslinked cysteine C94 was of much higher value. This may partially be due to the fact that Nash and Jonas (8) Relative changes in P-factors upon the addition of 500 mM sorbitol (A) and 2 M urea (B) for the pressure-assisted cold-denatured state.…”

“…The pressure was increased with a piston pump (HIP High Pressure Equipment Co., Frankfurt, Germany) and the samples were kept at 3.75 kbar at -13ºC. Under these conditions, HEWL appears to be in the pressure-assisted cold-denatured state (8). After the incubation time, the pressure of the vessel was slowly released, causing the sample fluid to freeze.…”

“…The difference of the averaged value and the single values was used as a measure of the error bar. k rc was calculated by the method of Bai et al (10) and the influence of the pressure was taken into account as indicated by Nash and Jonas (8). The k rc -values were not corrected for the influence of sorbitol and urea because the effects of co-solvents on the exchange rates can be neglected at low pH (12,13).…”

“…By this procedure, the pressure-assisted cold-denatured state of many proteins becomes accessible. The nature of the cold-unfolded state and its comparison to the heat-and pressure-induced unfolded state is still a matter of controversy, however, and only very little work has been carried out on pressure-assisted cold denaturation of proteins (3)(4)(5)(6)(7)(8). According to Marques et al (6), the reason for pressure-assisted cold denaturation lies in the inability of water molecules at the protein surface to arrange in the low density icelike structures which are favored with decreasing temperature.…”

The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure

“…Thus pressure destabilizes hydrophobic and electrostatic interactions in addition to eliminate cavities (20,30,31). A unique property of high pressure denaturation is the formation of partially folded or molten-globule states at equilibrium (32)(33)(34)(35). High pressure has been used successfully to denature and dissociate proteins, protein-DNA complexes, and virus particles (20, 25, 36 -39).…”