Protein Engineering, Vol. 15, No. 3, 185-192,
March 2002
© 2002 Oxford University Press
Molecular dynamics simulations as a tool for improving protein stability
1 Laboratory of Biochemistry and 2 Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands
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 
/ß-hydrolase fold main domain and a cap domain composed of five -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 52.5°C, whereas the Tm,app of the reduced mutant decreased by more than 8°C 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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Purmonen, J. Valjakka, K. Takkinen, T. Laitinen, and J. Rouvinen Molecular dynamics studies on the thermostability of family 11 xylanases Protein Eng. Des. Sel., November 1, 2007; 20(11): 551 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-J. Chen, I-S. Lee, C.-Y. Chen, and T.-H. Liao Biological functions of the disulfides in bovine pancreatic deoxyribonuclease Protein Sci., April 1, 2004; 13(4): 875 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vinayagam, G. Pugalenthi, R. Rajesh, and R. Sowdhamini DSDBASE: a consortium of native and modelled disulphide bonds in proteins Nucleic Acids Res., January 1, 2004; 32(90001): D200 - 202. [Abstract] [Full Text] [PDF]
|
|