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 and forms using temperature replica exchange molecular dynamics (REMD) data. We find a 14 K decrease in denaturation temperature for (406 K) azurin relative to that of (420 K), indicating a copper ion-induced additional thermal stability for azurin. The unfolding of azurin begins with the melting of α-helix and β-sheet V, similar to that of form. β-Sheets IV, VII, and VIII are comparatively more stable than other β-strands and melt at higher temperatures. Similar to azurin, the strong hydrophobic interactions among the apolar residues in the protein core is the key factor that renders high stability to 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 and 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 compared to the form, where β-sheet VI was relatively stable.