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

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• 作者：Rita Guzzi^a ; ^{rita.guzzi@fis.unical.it} ; Luigi Sportelli^a ; Sachiko Yanagisawa^b ; Chan Li^b ; Dorota Kostrz^b ; Christopher Dennison^b
• 关键词：Thermal unfolding ; Cupredoxins ; Kinetic stability ; DSC ; Optical absorption ; Fluorescence
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2012
• 期刊代码：116_00039861
• 类别：ch
• 出版时间：May, 2012
• 卷：521
• 期：1-2
• 页码：18-23
• 文件大小：777 K

摘要

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 掳C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1 掳C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at 鈭?00 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.