The role of salt bridges on the temperature adaptation of aqualysin I, a thermostable subtilisin-like proteinase

被引:29
|
作者
Jonsdottir, Lilja B. [1 ]
Ellertsson, Brynjar Oe. [1 ]
Invernizzi, Gaetano [3 ]
Magnusdottir, Manuela [1 ]
Thorbjarnardottir, Sigriour H. [2 ]
Papaleo, Elena [3 ]
Kristjansson, Magnus M. [1 ]
机构
[1] Univ Iceland, Inst Sci, Dept Biochem, IS-101 Reykjavik, Iceland
[2] Univ Iceland, Inst Biol, IS-101 Reykjavik, Iceland
[3] Univ Copenhagen, Dept Biol, Struct Biol & NMR Lab, DK-2200 Copenhagen, Denmark
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS | 2014年 / 1844卷 / 12期
关键词
Subtilisin-like; Salt bridges; Protein stability; Thermophilic; Protease; Molecular dynamics; ALKALINE SERINE-PROTEASE; COLD-ACTIVE ENZYME; ION-PAIRS; STABILITY; DYNAMICS; FLEXIBILITY; ELECTROSTATICS; DEHYDROGENASE; PURIFICATION; SIMULATIONS;
D O I
10.1016/j.bbapap.2014.08.011
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Differences in salt bridges are believed to be a structural hallmark of homologous enzymes from differently temperature-adapted organisms. Nevertheless, the role of salt bridges on structural stability is still controversial. While it is clear that most buried salt bridges can have a functional or structural role, the same cannot be firmly stated for ion pairs that are exposed on the protein surface. Salt bridges, found in X-ray structures, may not be stably formed in solution as a result of high flexibility or high desolvation penalty. More studies are thus needed to clarify the picture on salt bridges and temperature adaptation. We contribute here to this scenario by combining atomistic simulations and experimental mutagenesis of eight mutant variants of aqualysin I, a thermophilic subtilisin-like proteinase, in which the residues involved in salt bridges and not conserved in a psychrophilic homolog were systematically mutated. We evaluated the effects of those mutations on thermal stability and on the kinetic parameters. Overall, we show here that only few key charged residues involved in salt bridges really contribute to the enzyme thermal stability. This is especially true when they are organized in networks, as here attested by the D17N mutation, which has the most remarkable effect on stability. Other mutations had smaller effects on the properties of the enzyme indicating that most of the isolated salt bridges are not a distinctive trait related to the enhanced thermal stability of the thermophilic subtilase. (C) 2014 Elsevier B.V. All rights reserved.
引用
收藏
页码:2174 / 2181
页数:8
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