Prediction and experimental testing of Bacillus acidocaldarius thioredoxinstability

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R 82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius, per formed by a comparative molecular modelling technique using Escherichia col i thioredoxin as the reference protein. The mutants obtained by PCR strateg y were expressed in E. coli and then characterized. CD spectroscopy, spectr ofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of th e wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutan ts; in fact the T-m of the four proteins showed a decrease of about 15 degr ees C for the double and the truncated mutants, and a decrease of about 12 OC for the single mutants. A difference in the resistance of the proteins t o denaturants such as guanidine HCl and urea was revealed; the wild-type pr otein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stabilit y and confirm that simulation methods are able to direct protein engineerin g in site-directed mutagenesis.