Stabilization of tRNA (mG37) methyltransferase [TrmD] from Aquifex aeolicus by an intersubunit disulfide bond formation.
Recombinant Aquifex aeolicus TrmD protein has a Cys20-Cys20 disulfide bond between its two subunits. This was demonstrated by SDS-polyacrylamide gel analysis of wild-type enzyme and C20S mutant protein (in which the Cys20 residue is substituted by serine), in the absence or presence of various concentrations of dithiothreitol. Analytical gel-filtration chromatography revealed that the C20S mutant protein forms a dimer structure even though it is missing the disulfide bond. Western blotting analysis suggests that the Cys20-Cys20 disulfide bond is formed in native TrmD protein in living A. aeolicus cells. Incubation at 85 degrees C for 20 min caused the precipitation of more than half of the C20S protein, while more than 70% of the wild-type enzyme was soluble at that temperature. This assay clearly demonstrates that the disulfide bond enhances the protein stability at 85 degrees C. A kinetic assay showed that the methyl-transfer activity of the C20S mutant protein was slightly less than that of the wild-type enzyme at 70 degrees C. Comparison of the CD-spectra of wild-type and C20S proteins reveals that some of the alpha-helices in the C20S mutant protein are less tightly packed than those of the wild-type enzyme at 70 degrees C.
Toyooka
Takashi
T
Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime, Japan.
Awai
Takako
T
Kanai
Tamotsu
T
Imanaka
Tadayuki
T
Hori
Hiroyuki
H
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D012359:tRNA Methyltransferases / Q000737:chemistry*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012359:tRNA Methyltransferases; D003545:Cysteine