title: Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.
abstract: 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.
authors.affiliation: Department of Chemistry, Physical Chemistry I, University of Dortmund, Dortmund, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Vogtt
authors.forename: R
authors.initials: R
authors.lastname: Winter
mesh_terms: 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
chemical_list: D012997:Solvents; D013012:Sorbitol; D006859:Hydrogen; D014508:Urea; D009113:Muramidase