Unraveling the unfolding mechanism of pseudoazurin: Insights into stabilizing cupredoxin fold as a common domain of Cu-containing proteins

Understanding protein unfolding mechanisms is crucial for comprehending protein-folding related diseases, developing diagnostic methods, and designing proteins with desired stability for medicinal or industrial applications. However, investigating structures at atomic resolution is often difficult due to the flexibility and transiency of unfolding intermediate states. Pseudoazurin (PAz) is a well-characterized simple cupredoxin composed of a small polypeptide (124 amino acids) and a single metal cofactor (Cu²⁺), making it suitable to study the unfolding mechanism. In this study, combining the merits of structure determination by small-angle neutron scattering (SANS) and molecular dynamics (MD) simulations enabled us to access the details of the unfolding mechanism. The unfolding of PAz proceeds through a two-step mechanism involving the "native", "open-domain", and "random-coil" states. Several amino acid residues at the vicinity of Cu²⁺ ion are involved in the structural transitions, where the interactions among these residues are important in controlling the stability of PAz. These findings may be applicable to stabilizing metalloproteins with cupredoxin domain structures.