title: Exploring the protease mediated conformational stability in a trypsin inhibitor from Archidendron ellipticum seeds.
abstract: A Kunitz proteinase inhibitor from Archidendron ellipticum seeds (AeTI) was purified and complexed with bovine trypsin and chymotrypsin. The stoichiometric stability of AeTI with its interacting proteinases was then investigated using spectrophotometric, size exclusion chromatography (HPLC system), Western blotting and circular dichroism (CD) studies. All the methods were remarkably similar in revealing the preference of trypsin over chymotrypsin by AeTI for complex formation. Both Western blotting as well as spectrophotometry based assays for competition experiments indicated that trypsin displaces chymotrypsin from a previously formed AeTI-chymotrypsin complex. Chemical modification of lysine and arginine by TNBS and CHD treatments, respectively, suggested a lysine as the active site residue and also indicated the presence of a single protease-binding site for AeTI. CD of native AeTI showed a sharp minimum at 200 nm and deconvolution of the CD spectra revealed it to be an unordered protein possessing high beta-sheet content. Complex formation of AeTI with trypsin induces a fractional switchover of its unordered structure towards the beta-sheet fraction but lacked any such conversion in the presence of chymotrypsin. Prolonged exposure of excess trypsin generates conformational modifications both in the secondary and the tertiary structures.
authors.affiliation: Department of Botany, University of Delhi, Delhi 110007, India. ady1111@rediffmail.com
authors.forename: A
authors.initials: A
authors.lastname: Bhattacharyya
authors.forename: C R
authors.initials: CR
authors.lastname: Babu
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D002942:Circular Dichroism; D007887:Fabaceae / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry*
chemical_list: D046912:Multiprotein Complexes; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D014357:Trypsin