Molecular dynamics simulations as a tool for improving protein stability

被引:169
作者
Pikkemaat, MG
Linssen, ABM
Berendsen, HJC
Janssen, DB
机构
[1] Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Biochem Lab, NL-9747 AG Groningen, Netherlands
[2] Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Biophys Chem Lab, NL-9747 AG Groningen, Netherlands
来源
PROTEIN ENGINEERING | 2002年 / 15卷 / 03期
关键词
disulfide bond engineering; flexible region; haloalkane dehalogenase; molecular dynamics simulation; protein stability;
D O I
10.1093/protein/15.3.185
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
Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5degreesC, whereas the T-m,T-app of the reduced mutant decreased by more than 8degreesC compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.
引用
收藏
页码:185 / 192
页数:8
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