Unraveling the Molecular Origins of Exceptional Thermal Stability and Anti Cancer Properties of Metalloprotein Azurin

Abstract

Metalloproteins are a diverse class of proteins that incorporate one or more metal ions into their structure, crucially influencing their function within biological systems. Although metalloproteins make up a substantial portion of the proteome, understanding their dynamics, folding and unfolding processes, and the influence of metal cofactors on their stability continues to pose major challenges in the field. Also assessing the effect of metal cofactors on protein dynamics remains a fundamental challenge in the field. Azurin is one such metalloprotein containing a copper cofactor that is involved in electron transfer processes and exhibits very high thermal stability. Temperatures more than 80 °C are required for its irreversible denaturation. In our study, we probe the microscopic details for the high structural stability of this protein using an advanced sampling molecular dynamics technique called replica exchange molecular dynamics simulations (REMD). Our studies show that the unfolding of azurin begins with the melting of the and#120572;-helix region. The adjacent and#120573;-sheets, and#120573;-sheet V and and#120573;-sheet II, carry forward the melting process. Their partial melting results in enhanced interaction of water molecules with the protein. Gradually other and#120573;-sheets are also dislocated from their stiff and#120573;-barrel arrangement and melted. We found that the major factor contributing to the high stability of this protein is the hydrophobic interactions among a large number of non-polar and aromatic residues on the and#120573;-sheets of azurin, which are enhanced upon forming a collapsed core. To gain a comprehensive understanding of the intricate thermal unfolding pathways of azurin and the influence of copper on metalloprotein stability, it is essential to conduct a direct comparison of protein unfolding with and without the presence of bound copper metal ion. In this study, we observed a 14 K decrease in the denaturation temperature of apo azurin (without copper ion) compared to the holo form (with copper ion). Similar to holo azurin, the mel