1. A triple mutant (YI4F/Y30F/Y55F) of ketosteroid isomerase is more stable than its wild type through hydrophobic interaction as well as aromatic-aromatic interaction.
- Author
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Hyung Jin Cha, Do Soo Jang, Han Seop Shin, and Kwan Yong Choi
- Subjects
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ISOMERASES , *AROMATICITY , *STEROIDS , *KETONES , *CHEMICAL bonds , *GENETIC mutation - Abstract
One of important issues in protein science is to increase the protein stability. Utilizing a ketosteroid isomerase (KSI) as a model system, we constructed a mutant KSI which has a higher stability than its WT. We focused on the role of aromatic moieties of three tyrosine residues, Tyr14, Tyr55, and Tyr30, which constitute a hydrogen bond network in KSI. Therefore, we made a series of mutants which could substitute tyrosine with phenylalanine to remove the hydroxyl group in the tyrosine residue. We measured the free-energy change for unfolding using urea as a denaturant. We investigated the interaction among these aromatic residues for stability using the double mutant cycle analysis. We found that additional mutations on Y14F, Y55F, and Y14F/Y55F, respectively, by replacement of tyrosine at the position 30 with phenylalanine increased the stability ca. 4.0 kcal/mol. Especially the triple mutant KSI (Y14F/Y30F/Y55F) was more stable 2 kcal/mol than wild type KSI. Our structural analyses suggests that the enhanced stability of the triple mutant should be due to increased both hydrophobic interaction and aromatic-aromatic interaction upon the mutation [ABSTRACT FROM AUTHOR]
- Published
- 2007