Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Abstract

Unfolded and partially folded states of proteins play a crucial role in the formation of fibril aggregates in human neurodegenerative diseases. Some disease-associated proteins adopt well-folded structures in their native states while others belong to the class of intrinsically unstructured proteins. The conversion of a soluble form of a protein to the aggregated state is typically related to the transient formation of unfolded or partially folded states for both classes of proteins. In many protein-folding diseases, single-point mutations, often inherited, are linked to specific disease forms but the structural code for the onset of the disease is still unclear. Single-point mutations do not significantly change the structure of the native state. Their impact on the structural and dynamic characteristics of the unfolded or intrinsically unstructured state ensemble remains a key unsolved question in structural biology. One of the approximately 20 amyloid diseases known to date is associated with the deposition of amyloid plaques from single-point mutants (I56T, F57I, I59T, W64R, and D67H) of human lysozyme (hLys) in various organs. Given the remarkable high stability of this protein family, the discovery of protein-folding diseases associated with hLys came as a surprise. Initial studies revealed that the native-state structures of wildtype (wt) and amyloidogenic mutants of hLys are highly similar. Characterization of the thermal unfolding at pH 1.2 using CD spectroscopy, differential scanning calorimetry (DSC), and NMR spectroscopy showed that unfolding proceeds through loss of tertiary structure, resulting in a premolten globule state, which unfolds further as the temperature increases. The b domain and the adjacent helix 3 (residues 90–100) unfold first. The same region is destabilized in the mutants as shown by hydrogen exchange, leading to lower thermal stability. This decrease in stability suggests that the relative stabilities of the folded and unfolded states are important in the process of amyloid formation. In fact, amyloid fibrils are formed fastest when the protein is incubated at very low pH (1.2) and at temperatures near the midpoint of thermal unfolding, thus at a temperature at which folded as well as molten globule and premolten globule states, referred to here as unfolded states, are populated. For a better understanding of the mechanism leading to the formation of amyloid diseases, both folded and unfolded states therefore need to be characterized. In contrast to the folded state of hLys, little is known about the residual structure and dynamics of its nonnative unfolded state. Heteronuclear NMR spectroscopy permits the characterization of not only the structure but also the dynamics of unfolded nonnative protein ensembles at atomic resolution. To highlight the remarkable influence of the mutants on the structure and dynamics in unfolded hLys, we first compare the unfolded states of the hen egg white lysozyme (hewl)and hLys to highlight the intrinsic higher aggregation propensity of hLys relative to that of hewl, and then discuss how two amyloidogenic mutants modulate the