Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

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 ß-sheet V, similar to that of holo form. ß-Sheets IV, VII, and VIII are comparatively more stable than other ß-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 ß-sheets V and VI from the protein core is another distinction we observe for apo compared to the holo form, where ß-sheet VI was relatively stable.

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 ß-sheets II and V. This is followed by the melting of other ß-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 ß-sheets by looking into their amino acid sequence shows that ß-sheets with charged side chains on the surface melt fast compared to others. The ß-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 ß-sheets III, IV, and VII deviate less from their initial structure at the transition temperature.