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EnglishThe role played by the a-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 (HNMR)-H-1, 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 a-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 a-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 a-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.
The role played by the alfa-helix in the unfolding pathway and stability of azurin: Switching between hierarchic and nonhierarchic folding / MANETTO D., G; Grasso, Dm; Milardi, D; Pappalardo, M; Guzzi, R; Sportelli, Luigi; VERBEERT M., P; CANTERS G., W; LA ROSA, C.. - In: CHEMBIOCHEM. - ISSN 1439-4227. - 8(2007), pp. 1941-1949.
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| Titolo: | The role played by the alfa-helix in the unfolding pathway and stability of azurin: Switching between hierarchic and nonhierarchic folding | |
| Autori: | ||
| Data di pubblicazione: | 2007 | |
| Rivista: | ||
| Citazione: | The role played by the alfa-helix in the unfolding pathway and stability of azurin: Switching between hierarchic and nonhierarchic folding / MANETTO D., G; Grasso, Dm; Milardi, D; Pappalardo, M; Guzzi, R; Sportelli, Luigi; VERBEERT M., P; CANTERS G., W; LA ROSA, C.. - In: CHEMBIOCHEM. - ISSN 1439-4227. - 8(2007), pp. 1941-1949. | |
| Handle: | http://hdl.handle.net/20.500.11770/138909 | |
| Appare nelle tipologie: | 1.1 Articolo in rivista |
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