Premium
The Role Played by the α‐Helix in the Unfolding Pathway and Stability of Azurin: Switching Between Hierarchic and Nonhierarchic Folding
Author(s) -
Manetto Gaetano D.,
Grasso Domenico M.,
Milardi Danilo,
Pappalardo Matteo,
Guzzi Rita,
Sportelli Luigi,
Verbeet Martin P.,
Canters Gerard W.,
La Rosa Carmelo
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700214
Subject(s) - azurin , helix (gastropod) , chemistry , folding (dsp implementation) , crystallography , protein folding , circular dichroism , electron paramagnetic resonance , biophysics , turn (biochemistry) , electron transfer , photochemistry , biochemistry , nuclear magnetic resonance , biology , ecology , physics , snail , electrical engineering , engineering
The role played by the α‐helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix‐depleted azurin variant produced by sitedirected 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 α‐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 α‐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 β‐sheet cupredoxins, the results as a whole support the hypothesis that the presence of the α‐helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved β ‐sheet core provides a scaffold for cooperative folding of the wild‐type protein.