Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?
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.
Bonander
N
N
Department of Biochemistry and Biophysics, Lundberg Institute, Göteborg University and Chalmers University of Technology, Sweden.
Leckner
J
J
Guo
H
H
Karlsson
B G
BG
Sjölin
L
L
D001400:Azurin / Q000737:chemistry* / Q000235:genetics*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014466:Ultraviolet Rays; D015032:Zinc / Q000494:pharmacology
D004220:Disulfides; D001400:Azurin; D015032:Zinc; D019791:Guanidine