title: The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding.
abstract: 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.
authors.affiliation: Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 (CT), Italy.
authors.forename: Gaetano D
authors.initials: GD
authors.lastname: Manetto
authors.forename: Domenico M
authors.initials: DM
authors.lastname: Grasso
authors.forename: Danilo
authors.initials: D
authors.lastname: Milardi
authors.forename: Matteo
authors.initials: M
authors.lastname: Pappalardo
authors.forename: Rita
authors.initials: R
authors.lastname: Guzzi
authors.forename: Luigi
authors.initials: L
authors.lastname: Sportelli
authors.forename: Martin P
authors.initials: MP
authors.lastname: Verbeet
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Canters
authors.forename: Carmelo
authors.initials: C
authors.lastname: La Rosa
mesh_terms: 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
chemical_list: D001400:Azurin