title: The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa.
abstract: The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7°C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1°C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at ∼600nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins.
authors.affiliation: Molecular Biophysics Laboratory and CNISM Unit, Department of Physics, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy. rita.guzzi@fis.unical.it
authors.forename: Rita
authors.initials: R
authors.lastname: Guzzi
authors.forename: Luigi
authors.initials: L
authors.lastname: Sportelli
authors.forename: Sachiko
authors.initials: S
authors.lastname: Yanagisawa
authors.forename: Chan
authors.initials: C
authors.lastname: Li
authors.forename: Dorota
authors.initials: D
authors.lastname: Kostrz
authors.forename: Christopher
authors.initials: C
authors.lastname: Dennison
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics; D056811:Unfolded Protein Response
chemical_list: D001400:Azurin