Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability.
In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, 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, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the 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 mutants; in fact the Tm of the four proteins showed a decrease of about 15 degrees C for the double and the truncated mutants, and a decrease of about 12 degrees C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.
Pedone
E
E
Dipartimento di Chimica Organica e Biologica, Università degli Studi di Napoli 'Federico II', via Mezzocannone 16, 80134 Naples, Italy.
Cannio
R
R
Saviano
M
M
Rossi
M
M
Bartolucci
S
S
D001407:Bacillus / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D009154:Mutation; D010078:Oxazines / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D010078:Oxazines; D013879:Thioredoxins; C044808:nile red