Crystal structure of the disulfide bond‐deficient azurin mutant C3A/C26A

Azurin has a β‐barrel fold comprising eight β‐strands and one α helix. A disulfide bond between residues 3 and 26 connects the N‐termini of β strands β1 and β3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild‐type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 Å resolution and, in comparison to the wild‐type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge‐transfer band at a temperature 10–22 °C lower than the wild‐type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3  m GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is −24.9 kJ·mol −1 , a destabilization of ≈ 20 kJ·mol −1 compared to the wild‐type Zn 2+ ‐protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild‐type Zn 2+ ‐azurin is not observed for the disulfide‐deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild‐type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.