The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

Protein Sci. 2004 Aug;13(8):2184-95. doi: 10.1110/ps.04769004.

Abstract

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence / genetics
  • Bacillus subtilis / enzymology*
  • Bacillus subtilis / genetics
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Dimerization
  • Geobacillus stearothermophilus / enzymology*
  • Geobacillus stearothermophilus / genetics
  • Glycine Hydroxymethyltransferase / chemistry*
  • Glycine Hydroxymethyltransferase / genetics
  • Guanidine / chemistry
  • Molecular Sequence Data
  • Protein Denaturation / genetics
  • Protein Folding*
  • Protein Structure, Tertiary / genetics
  • Sequence Homology, Amino Acid*

Substances

  • Bacterial Proteins
  • Glycine Hydroxymethyltransferase
  • Guanidine