Unfolding of hen egg-white lysozyme - is there a unique starting point?

Proteins can be unfolded in a regulated way by use of different strategies, which include changes of temperature or pressure, addition of chemical denaturants to and/or alteration of the pH value of the solution in which the protein is immersed. It would be of interest to know if the onset of unfolding by these different methods has a common starting point, since protein misfolding that is preceded by unfolding is associated with a number of neurological diseases. We have used the technique of X-ray crystallography to identify onset of unfolding of hen egg white lysozyme (HEWL), which is a relatively small two-domain enzyme with optimal activity at pH 4.3. Here we report high-resolution structures of the protein at the extreme pH values of 2.0 and 12.0. We find that while the overall structure of the protein is native-like, the side chain conformations of two beta-sheet residues ASN46 and ASN59 near the active site are significantly altered. As a consequence, hydrogen bonds involving these side chains are lost suggesting loss of stability. Interestingly, identical conformational change and loss of hydrogen bonds was also observed in structures of HEWL exposed either to denaturants or to higher temperatures. We suggest that this common mode of weakening of the beta-sheet structure through ASN59 is the starting point in the unfolding of HEWL. This observation is in contrast to earlier suggestion that disruption in the hydrophobic interface between alpha and beta domains is the starting point of unfolding in HEWL.