Role of Metal Cofactor in Enhanced Thermal Stability of Azurin

Metal cofactors are critical centersfor different biochemicalprocesses of metalloproteins, and often, this metal coordination rendersadditional structural stability. In this study, we explore the additionalstability conferred by the copper ion on azurin by analyzing boththe apo and holo forms using temperaturereplica exchange molecular dynamics (REMD) data. We find a 14 K decreasein denaturation temperature for apo (406 K) azurinrelative to that of holo (420 K), indicating a copperion-induced additional thermal stability for holo azurin. The unfolding of apo azurin begins withthe melting of alpha-helix and beta-sheet V, similar to thatof holo form. beta-Sheets IV, VII, and VIII arecomparatively more stable than other beta-strands and melt at highertemperatures. Similar to holo azurin, the stronghydrophobic interactions among the apolar residues in the proteincore is the key factor that renders high stability to apo protein as well. We construct free energy surfaces at differenttemperatures to capture the major conformations along the unfoldingbasins of the protein. Using contact maps from different basins weshow the changes in the interaction between different residues alongthe unfolding pathway. Furthermore, we compare the C alpha root-mean-squarefluctuations (C alpha-RMSF) and B-factor of all residues of apo and holo forms to understand the flexibilityof different regions. The concerted displacement of alpha-helixand beta-sheets V and VI from the protein core is another distinctionwe observe for apo compared to the holo form, where beta-sheet VI was relatively stable.

Automated summary

In this study, the additional stability conferred by the copper ion on azurin was explored using REMD data. It was found that holo azurin had a higher denaturation temperature compared to apo azurin, indicating an additional thermal stability induced by the copper ion. The unfolding pathway of apo azurin was similar to that of holo azurin, with certain beta-sheets being more stable. The strong hydrophobic interactions among apolar residues in the protein core contributed to the high stability of both apo and holo forms.