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.
Muricken
Deepa G
DG
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific and Industrial Research (CSIR) Mysore, 570-020, India.
Gowda
Lalitha R
LR
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