Molecular Insight into the High Thermal Stability of Metalloprotein Azurin.

We investigate the events characterizing the steps of the unfolding pathway of blue copper metalloprotein azurin using replica exchange molecular dynamics (REMD). Our studies show that the unfolding of azurin begins with the melting of α-helix and β-sheets II and V. This is followed by the melting of other β-sheets and the exposure of hydrophobic protein core to the solvent, resulting in disruptions of its tertiary structure. Free energy surfaces constructed at different temperatures portray different basins that signify the stability of different melted structures in the unfolding process. The contact maps at different temperatures reveal that the strong hydrophobic interaction within the core of the protein is the vital force that renders high stability to this protein. Analysis of the individual β-sheets by looking into their amino acid sequence shows that β-sheets with charged side chains on the surface melt fast compared to others. The β-barrel of azurin is able to dynamically rearrange, and it helps the protein to preserve its hydrophobic core, holding back the native topology from melting fast. B-factor analysis shows that residues of β-sheets III, IV, and VII deviate less from their initial structure at the transition temperature.