Cavity hydration as a gateway to unfolding: An NMR study of hen lysozyme at high pressure and low temperature

We have used low temperatures (down to − 20 °C) and high pressures (up to 2000 bar) to populate low-lying excited state conformers of hen lysozyme, and have analyzed their structures site-specifically using 15N/1H two-dimensional HSQC NMR spectroscopy. The resonances of a number of residues were found to be selectively broadened, as the temperature was lowered at a pressure of 2000 bar. The resulting disappearance of cross-peaks includes those of residues in the β-domain of the protein and the cleft between the β- and α-domains, both located close to water-containing cavities. The results indicate that low-lying excited state conformers of hen lysozyme are characterized by slowly fluctuating local conformations around these cavities, attributed to the opportunities for water molecules to penetrate into the cavities. Furthermore, we have found that these water-containing cavities are conserved in similar positions in lysozymes from a range of different biological species, indicating that they are a common evolutionary feature of this family of enzymes.

Graphical AbstractResearch highlights► We used 15N/1H two-dimensional NMR at high pressure and low temperature to detect pre-hydrated low-lying excited states within the folded manifold of hen lysozyme. ► Cavity-linked heterogeneously hydrated intermediates are detected predominantly in the β-domain. ► The β-hydrated state is considered to precede the α-domain intermediate found previously in the unfolding process of hen lysozyme.