Aneta Szymańska

Human cystatin C (hCC), a small secretory protein, has gained attention beyond its classical role as a cysteine protease inhibitor owing to its potential involvement in neurodegenerative disorders. This study investigates the interaction between copper(II) ions [Cu(II)] and hCC, specifically targeting histidine residues known to participate in metal binding. Through various analytical techniques, including mutagenesis, circular dichroism, fluorescence assays, gel filtration chromatography, and electron microscopy, we evaluated the impact of Cu(II) ions on the structure and oligomerization of hCC. The results show that Cu(II) does not influence the secondary and tertiary structure of the studied hCC variants but affects their stability. To explore the Cu(II)‐binding site, nuclear magnetic resonance (NMR) and X‐ray studies were conducted. NMR experiments revealed notable changes in signal intensities and linewidths within the region 86 His‐Asp‐Gln‐Pro‐His 90 , suggesting its involvement in Cu(II) coordination. Both histidine residues from this fragment were found to serve as a primary anchor of Cu(II) in solution, depending on the structural context and the presence of other Cu(II)‐binding agents. The presence of Cu(II) led to significant destabilization and altered thermal stability of the wild‐type and H90A variant, confirming differentiation between His residues in Cu(II) binding. In conclusion, this study provides valuable insights into the interaction between Cu(II) and hCC, elucidating the impact of copper ions on protein stability and identifying potential Cu(II)‐binding residues. Understanding these interactions enhances our knowledge of the role of copper in neurodegenerative disorders and may facilitate the development of therapeutic strategies targeting copper‐mediated processes in protein aggregation and associated pathologies.