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The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa

Lookup NU author(s): Sachiko Yanagisawa, Dorota Kostrz, Professor Christopher Dennison


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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 degrees C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1 degrees C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at similar to 600 nm 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. (C) 2012 Elsevier Inc. All rights reserved.

Publication metadata

Author(s): Guzzi R, Sportelli L, Yanagisawa S, Li C, Kostrz D, Dennison C

Publication type: Article

Publication status: Published

Journal: Archives of Biochemistry and Biophysics

Year: 2012

Volume: 521

Issue: 1-2

Pages: 18-23

Print publication date: 14/03/2012

ISSN (print): 0003-9861

ISSN (electronic): 1096-0384

Publisher: Academic Press


DOI: 10.1016/


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Funder referenceFunder name
University of Calabria
BB/C504519Biotechnology and Biological Sciences Research Council
MCRTN-035649-EdRoxEuropean Community through the EdRox Marie Curie Research Training Network