Functional expression of horsegram (Dolichos biflorus) Bowman-Birk inhibitor and its self-association.
Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, native to Southeast Asia and tropical Africa, contains multiple forms of Bowman-Birk inhibitors. The major Bowman-Birk inhibitor from horsegram (HGI-III) was cloned and functionally expressed in Escherichiacoli (rHGI), which moved as a dimer in solution similar to the natural inhibitor. The biochemical characterization of rHGI also points to its close resemblance with HGI-III not only in its structure but also in its inhibitory characteristics. To explore the electrostatic interactions involved in the dimerization, a site-directed mutagenesis approach was used. The role of reactive site residue K24 and the C-terminal Asp in the structure and stability of the dimer was accomplished by mutating K24 and D75/76. The mutants produced in this study confirm that the self-association of HGI-III is indeed due to the electrostatic interaction between K24 of one monomer and D75/76 of the second monomer, in agreement with our previous data. The functional expression of a Bowman-Birk inhibitor minus a fusion tag serves as a platform to study the structural and functional effects of the special pattern of seven conserved disulphide bridges.
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific and Industrial Research (CSIR) Mysore, 570-020, India.
Deepa G
DG
Muricken
Lalitha R
LR
Gowda
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002417:Cattle; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D031294:Dolichos; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012639:Seeds / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry*
D004220:Disulfides; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D001224:Aspartic Acid