The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding.
The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.
Manetto
Gaetano D
GD
Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 (CT), Italy.
Grasso
Domenico M
DM
Milardi
Danilo
D
Pappalardo
Matteo
M
Guzzi
Rita
R
Sportelli
Luigi
L
Verbeet
Martin P
MP
Canters
Gerard W
GW
La Rosa
Carmelo
C
D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012015:Reference Standards; D013696:Temperature
D001400:Azurin