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 (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). 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 Pfactors 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.

Protein Denaturation on p-T Axes – Thermodynamics and Analysis

“…A number of experimental works were published on the pressure and temperature unfolding of lysozyme (Sasahara et al 2001;Refaee et al 2003;Vogtt and Winter 2005;Maeno et al 2009;Smeller 2009). Most often the hen egg white (HEW) lysozyme was investigated, since it is commercially available.…”

“…Winter's group studied the heat denatured and the pressure assisted cold denaturated state of HEW lysozyme using COSY proton NMR (Vogtt and Winter 2005). They measured the hydrogen-deuterium exchange rate and determined the protection factors for several amino acid residues.…”

Highlights of the 3rd International Conference on High Pressure Bioscience and Biotechnology

“…This was interpreted to indicate that the presence of membranes stabilized the protein complex due to the protein confinement on its surface, therefore pointing to a role of a reduced volume in the lateral association between membrane proteins. This presentation was followed by that of Prof. Roland Winter (University of Dortmund, Germany), Exploring the Con- (13). The flip side of protein folding and unfolding by high hydrostatic pressure and temperature was also explored from a physicochemical point of view using membrane proteins and soluble proteins as models, and a threedimensional free energy surface diagram for protein stability was presented (13).…”

“…This presentation was followed by that of Prof. Roland Winter (University of Dortmund, Germany), Exploring the Con- (13). The flip side of protein folding and unfolding by high hydrostatic pressure and temperature was also explored from a physicochemical point of view using membrane proteins and soluble proteins as models, and a threedimensional free energy surface diagram for protein stability was presented (13). Prof. Jerson L. Silva presented New Insights into the Mechanism of Protein Folding and Misfolding Derived from Pressure Studies, with an emphasis on the prion diseases (14).…”

Low crowding agent concentration destabilizes against pressure unfolding

“…Bovine serum albumin (BSA) is an average size protein with a 66 kDa molar mass, but lysozyme is considerably smaller (14 kDa). These proteins have been well characterized by several spectroscopic techniques, and the pressure behavior of these proteins in absence of crowding agents is also known [32][33][34][35].…”