Role of Metal Cofactor in Enhanced Thermal Stability of Azurin

Metal cofactors are critical centers for different biochemical processes of metalloproteins, and often, this metal coordination renders additional structural stability. In this study, we explore the additional stability conferred by the copper ion on azurin by analyzing both the apo and holo forms using temperature replica exchange molecular dynamics (REMD) data. We find a 14 K decrease in denaturation temperature for apo (406 K) azurin relative to that of holo (420 K), indicating a copper ion-induced additional thermal stability for holo azurin. The unfolding of apo azurin begins with the melting of α-helix and β-sheet V, similar to that of holo form. β-Sheets IV, VII, and VIII are comparatively more stable than other β-strands and melt at higher temperatures. Similar to holo azurin, the strong hydrophobic interactions among the apolar residues in the protein core is the key factor that renders high stability to apo protein as well. We construct free energy surfaces at different temperatures to capture the major conformations along the unfolding basins of the protein. Using contact maps from different basins we show the changes in the interaction between different residues along the unfolding pathway. Furthermore, we compare the Cα root-mean-square fluctuations (Cα-RMSF) and B-factor of all residues of apo and holo forms to understand the flexibility of different regions. The concerted displacement of α-helix and β-sheets V and VI from the protein core is another distinction we observe for apo compared to the holo form, where β-sheet VI was relatively stable.