Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques
✍ Scribed by R. Wynn; F. M. Richards
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2008
- Tongue
- English
- Weight
- 899 KB
- Volume
- 2
- Category
- Article
- ISSN
- 0961-8368
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1‐n‐propane, 1‐n‐butane, and 1‐n‐pentane thiols. These mutants all show native‐like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]~1/2~, and the free energy of unfolding at zero denaturant concentration, δ__G__, give inverse orders of stability. This effect is due to changes in m, the dependence of δ__G__~unfolding~^0^ on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.