title: Site-directed mutagenesis to identify key residues in structure-function relationship of winged bean chymotrypsin-trypsin inhibitor and 3-D structure prediction.
abstract: Winged bean chymotrypsin trypsin inhibitor (WbCTI) is a Kunitz type serine protease inhibitor that inhibits both trypsin and chymotrypsin at 1:1 molar ratio. Site-directed mutagenesis study was employed to generate two mutants of WbCTI, with an aim to explore its dual inhibitory properties against the proteases. The mutants were expressed in Escherichia coli and, were purified to homogeneity using a single step immunoaffinity column. The two mutants, each containing a single mutation at the amino acid sequence positions of 63 and 64, were named as L63A and R64A, respectively. Purified L63A-WbCTI exhibited anti-trypsin activity with no anti-chymotrypsin activity whereas R64A-WbCTI could inhibit chymotrypsin but not trypsin. To investigate the binding interactions between the mutated forms of WbCTI with the putative proteases, binding studies were carried out using gel filtration chromatography which further confirmed the formation of enzyme-inhibitor complexes. Finally, 3D model structure of WbCTI was designed using computer simulations which further emphasize the roles of L63 and R64 residues for dual inhibitory characteristics of WbCTI.
authors.affiliation: Department of Molecular and Human Genetics, Indian Institute of Chemical Biology, Kolkata, India. sxr252@case.edu
authors.forename: Sanhita
authors.initials: S
authors.lastname: Roy
authors.forename: Chhabinath
authors.initials: C
authors.lastname: Mandal
authors.forename: Samir K
authors.initials: SK
authors.lastname: Dutta
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000378:metabolism; D007887:Fabaceae / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014361:Trypsin Inhibitors / Q000737:chemistry
chemical_list: D010940:Plant Proteins; D014361:Trypsin Inhibitors; C440588:WCI protein, Psophocarpus tetragonolobus; D002918:Chymotrypsin