The contribution of two disulfide bonds in the trypsin binding domain of horsegram (Dolichos biflorus) Bowman-Birk inhibitor to thermal stability and functionality.

The major Bowman-Birk inhibitor (BBIs) of horsegram (Dolichos biflorus) HGI-III, contains seven interweaving
disulfides and is extremely stable to high temperatures. The contributions of two disulfide bonds
in the trypsin domain to thermal stability and functionality were evaluated using disulfide deletion variants
of wild type protein. Thermal denaturation kinetics, differential scanning calorimetry and urea denaturation
studies indicate that the absence of either of the two disulfides destabilizes the protein
significantly. C20–C66 contributes substantially to both thermal stability and controls trypsin and chymotrypsin
inhibitor activity. These two disulfides act in synergy as deletion of both disulfides leads to
a complete loss of thermal stability. The data indicate that the two subdomains are not entirely independent
of each other. Long range interactions, between the domains are facilitated by C20–C66. The deletion
of the disulfide bonds also increased proteolytic susceptibility in a manner similar to the decreased thermal
stability. From this study of rHGI a prototype of legume BBIs in can be concluded that among the
array of seven evolutionarily conserved disulfide bonds, the disulfide C20–C66 that connects a residue
in the trypsin domain with a residue at the border of the same domain plays a dominant role in maintaining
functional and structural stability.