Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.
COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.
Vogtt
K
K
Department of Chemistry, Physical Chemistry I, University of Dortmund, Dortmund, Germany.
Winter
R
R
D000818:Animals; D002645:Chickens; D003080:Cold Temperature; D004529:Egg White; D006358:Hot Temperature; D006859:Hydrogen / Q000494:pharmacology; D006874:Hydrostatic Pressure; D008279:Magnetic Resonance Imaging / Q000379:methods; D009113:Muramidase / Q000737:chemistry / Q000187:drug effects*; D011489:Protein Denaturation / Q000187:drug effects; D012997:Solvents / Q000494:pharmacology*; D013012:Sorbitol / Q000494:pharmacology*; D014508:Urea / Q000494:pharmacology
D012997:Solvents; D013012:Sorbitol; D006859:Hydrogen; D014508:Urea; D009113:Muramidase