The role played by the a-helix in the unfolding pathway and stability of azurin: switching between hierarchic and non-hierarchic folding

The role played by the α-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying an helix-depleted azurin variant produced by site-directed mutagenesis. Protein structure was investigated by CD, 1D 1H-NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-Vis and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effect of the α-helix depletion on the thermal stability and the unfolding pathway of the protein was determined by DSC, UV-Vis and fluorescence measurements at increasing temperature. The results show that in the absence of the α-helix segment the overall protein structure is maintained, and that only the Cu site is slightly modified. By contrast, 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. If compared with previous studies concerning other small β-sheet cupredoxins, the whole of the results support the hypothesis that the presence of the -helix can switch the folding of azurin from a hierarchic to a non-hierarchic mechanism in which the highy conserved β-shet core provides a scaffold for cooperative folding of the wt protein.