Structural characterisation and comparison of the native and A-states of equine lysozyme.

This study examines the structural characteristics of equine lysozyme in its native state and in a partially folded state known as the A-state. The research utilised nuclear magnetic resonance (NMR) measurements and hydrogen exchange kinetics to study the behaviour of the protein in these different states and proposes a model for the A-state structure.

NMR Resonance Assignments

• The team used nuclear magnetic resonance (NMR) to assign resonances to 125 of the 129 residues of equine lysozyme. This assignment technique allowed the researchers to identify individual atoms within the protein, which is a key step in understanding its three-dimensional structure.
• The hydrogen exchange kinetics measurement for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state was also enabled by these assignments. Hydrogen exchange kinetics is a technique used to study the dynamics, stability, and folding of proteins. The slow exchange of some hydrogen atoms indicates that they are part of the stable, folded core of the protein.

Native Versus A-State

• The difference between the native and A-state of the protein is most evident in their level of order. The native state is highly ordered, with high hydrogen exchange protection factors up to 10(6), especially in regions participating in secondary structure elements like alpha-helices and beta-sheets.
• However, the A-state, which appears at low pH, is globally much less ordered but does not contain significant random coil structure, indicating some level of residual structure. The protection factors in this state are only up to 10(2) at 5 degrees Celsius, far lower than in the native state, indicating a higher level of dynamic motion and reactivity.

A-State Structure

• The findings, including the binding characteristics of a fluorescent probe, a differential hydrogen exchange rate, and spectral data, suggest that the A-state maintains some of the secondary structural elements, namely three out of four alpha-helices.
• Furthermore, side-chains of these residues form a hydrophobic cluster, including five aromatic residues. This suggests a complex structure in the A-state with a more ordered core and a less ordered but compact periphery, rather a simple random coil or molten globule.

Overall, the research reveals the dynamic differences between the different states of equine lysozyme and sheds some light on the structural characteristics of its low-pH A-state.