The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa
The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa
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DOI：
10.1016/j.abb.2012.03.007
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发表时间：
2012-05-01
影响因子：
3.9
通讯作者：
Dennison, Christopher
中科院分区：
文献类型：
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作者：
Guzzi, Rita;Sportelli, Luigi;Dennison, Christopher
铜网站和总体结构的天青蛋白（AZ）的变体，其中amicyanin（AMI）和质体蓝素（PC）的金属结合环已被引入，AZAMI和AZPC，分别是类似的AZ，而环构象类似于那些在天然蛋白质。为了评估这些环突变对稳定性的影响，通过差示扫描量热法、吸收光谱和荧光光谱研究了AZAMI和AZPC的热去折叠。这两种变体的量热曲线都呈现出由两个吸热峰和一个放热峰组成的复杂形状。AZ的单个吸热峰的最大吸收热的温度为82.7 ℃，而AZAMI和AZPC的最强吸热峰分别为74.9和68.1 ℃，与AMI和PC的值相当。使用在类似于600 nm处的吸光度和Trp发射的温度依赖性研究的变性也证明了两种环突变体的稳定性降低。天然和变性状态之间的热转变是不可逆的，扫描速率相关的，并符合两态不可逆模型。活性位点环的结构对铜氧还蛋白的动力学稳定性和去折叠途径具有显著影响。(C)2012 Elsevier Inc. All rights reserved.
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.