title: Characterization of pyridoxal 5'- phosphate-binding domain and folding intermediate of Bacillus subtilis serine hydroxymethyltransferase: an autonomous folding domain.
abstract: The pyridoxal-5'-phosphate-binding domain (PLPbd) of bsSHMT (Bacillus subtilis serine hydroxymethyltransferase) was cloned and over-expressed in Escherichia coli. The recombinant protein was solublized, refolded and purified from inclusion bodies by rapid mixing followed by ion exchange chromatography. Structural and functional studies suggested the native form of the domain, which obtained as a monomer and had similar secondary and tertiary structural properties as when present in the bsSHMT. The domain also binds to the PLP however with slightly lesser affinity than the native enzyme. GdmCl (guanidium chloride)-induced equilibrium unfolding of the recombinant PLP-binding domain showed a single monophasic transition which corresponds with the second phase transition of the GdmCl-induced unfolding of bsSHMT. The results indicate that PLPbd of bsSHMT is an independent domain, which attains its tertiary structure before the dimerization of partially folded monomer and behaves as a single cooperative unfolding unit under equilibrium conditions.
authors.affiliation: Division of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Delhi 110 054, India. anant.bhatt@indiatimes.com
authors.forename: Anant Narayan
authors.initials: AN
authors.lastname: Bhatt
authors.forename: Vinod
authors.initials: V
authors.lastname: Bhakuni
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase; D019791:Guanidine