1EZL

CRYSTAL STRUCTURE OF THE DISULPHIDE BOND-DEFICIENT AZURIN MUTANT C3A/C26A: HOW IMPORTANT IS THE S-S BOND FOR FOLDING AND STABILITY?

Summary for 1EZL

• Entry DOI: 10.2210/pdb1ezl/pdb
• Descriptor: AZURIN, COPPER (II) ION (3 entities in total)
• Functional Keywords: mutant, disulphide bond, protein folding, greek key fold, electron transport
• Biological source: Pseudomonas aeruginosa
• Cellular location: Periplasm: P00282
• Total number of polymer chains: 4
• Total formula weight: 55844.86

Authors

Bonander, N.,Leckner, J.,Guo, H.,Karlsson, B.G.,Sjolin, L. (deposition date: 2000-05-11, release date: 2000-08-09, Last modification date: 2024-02-07)

Primary citation

Bonander, N.,Leckner, J.,Guo, H.,Karlsson, B.G.,Sjolin, L.
Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?
Eur.J.Biochem., 267:4511-4519, 2000

PubMed Abstract: Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. 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 A 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 degrees 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 approximately 20 kJ.mol-1 compared to the wild-type Zn2+-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 Zn2+-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.

PubMed: 10880975

Experimental method

X-RAY DIFFRACTION (2 Å)