Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals.

The hydrogen/deuterium (H/D) exchange of main-chain amide hydrogens in the protein that denatured and refolded in deuterated solvent is considered to contain the traces of hydrogen bond cleavages or the exposure to solvent of the buried part of the protein during the denaturing and refolding (denaturing/refolding) processes. Here, we report the H/D exchange behaviors in hen egg-white lysozymes denatured under acidic conditions, basic conditions, and thermal conditions and then refolded in deuterated solvents, using crystallographic methods. The results indicate that the space containing the Trp28 side chain was hardly exposed to the solvent in acidic conditions, but exposed under basic or heated conditions. Moreover, the β-bridges between Tyr53 and Ile58 in strands β2 and β3, which are in a highly conserved region, show some tolerance to changes in pD. The results indicate that crystallographic method is one of the powerful tools to analyze the denaturing/refolding processes of proteins.