1ezl

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spinqualitylabelsall models 1ezl, resolution 2.0Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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

Overview

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.

About this Structure

1EZL is a Single protein structure of sequence from Pseudomonas aeruginosa with CU as ligand. Full crystallographic information is available from OCA.

Reference

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?, Bonander N, Leckner J, Guo H, Karlsson BG, Sjolin L, Eur J Biochem. 2000 Jul;267(14):4511-9. PMID:10880975

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