Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional (1)H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a T(m) of 76.0 ± 0.2°C and no appreciable loss of the secondary structure up to 85°C. This is in contrast to the coincident denaturation of both tertiary and secondary structures with T(m) values of 74.8 ± 0.4°C and 74.3 ± 0.7°C, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.