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Role of Metal Cofactor in Enhanced Thermal Stability of Azurin

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Summary: We used replica exchange molecular dynamics (REMD) to investigate the unfolding pathway of azurin, a blue copper metalloprotein. Our findings suggest that the unfolding of azurin starts with the melting of alpha-helix and beta-sheets II and V, followed by the melting of other beta-sheets and the exposure of the hydrophobic protein core to the solvent. Different temperature-dependent free energy surfaces indicate the stability of various melted structures during the unfolding process. Contact maps at different temperatures reveal that strong hydrophobic interactions within the protein core contribute to its high stability. Analysis of individual beta-sheets shows that beta-sheets with charged side chains on the surface melt faster compared to others. The beta-barrel structure of azurin can dynamically rearrange, preserving the hydrophobic core and slowing down the melting of the native topology. B-factor analysis indicates that residues of beta-sheets III, IV, and VII undergo less deviation from their initial structure at the transition temperature.

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