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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

¹ Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India
² Department of Biochemistry, Dr. R.M.L. Avadh University, Faizabad 224001, India

(RECEIVED March 24, 2004; FINAL REVISION April 27, 2004; ACCEPTED May 8, 2004)

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share ~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.

Keywords: serine hydroxymethyltransferase; chimera; thermophilic; mesophilic; unfolding; guanidine hydrochloride; dissociation; cooperative unfolding

Abbreviations: SHMT, serine hydroxymethyltransferase • PLP, pyridoxal-5'-phosphate • PyP, pyridoxamine-P • SEC, size exclusion chromatography • ESI-MS, electron spray ionization mass spectroscopy • Tm, midpoint of thermal denaturation • GdmCl, guanidium chloride.

Reprint requests to: Vinod Bhakuni, Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India; e-mail: bhakuniv{at}rediffmail.com; fax: 91-522-223405.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04769004.

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