A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant.
The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.
Guzzi
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R
Dipartimento di Fisica e Unità INFM, Laboratorio di Biofisica Molecolare, Università della Calabria, 87030 Rende (CS), Italy.
Sportelli
L
L
La Rosa
C
C
Milardi
D
D
Grasso
D
D
Verbeet
M P
MP
Canters
G W
GW
D000409:Alanine / Q000737:chemistry; D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics
D017931:DNA Primers; D001400:Azurin; D003545:Cysteine; D000409:Alanine