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Molecular Engineering of the Horsegram (Dolichos biflorus) Seed Bowman-Birk Inhibitor: Implications of the disulfide framework on functionality.

Vinod, Kumar (2014) Molecular Engineering of the Horsegram (Dolichos biflorus) Seed Bowman-Birk Inhibitor: Implications of the disulfide framework on functionality. Doctoral thesis, University of Mysore.

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Abstract

The Bowman-Birk inhibitors (BBIs) are a group of small proteinproteinase inhibitors present in most legumes. These inhibitors play a key role in plant arsenal against insect herbivory and combating proteinases of pests and pathogens. The seeds of the legume horsegram (Dolichos biflorus), a protein rich pulse (bean), contain multiple forms of BBIs. The major inhibitor HGI-III contains seven interweaving disulfides and is extremely stable to high temperatures and acid pH. A soluble HGI-III (rHGI) with the native N-terminus was produced using a pTWIN IMPACTTM purification system. A fusion protein consisting of a chitin binding domain (CBD), a modified Ssp dnaB mini intein (dnaB) and HGI-III was expressed as a soluble protein in E. coli. After chitin bead affinity purification rHGI was separated from its fusion partner (CBD-dnaB) by a pH shift induced self-cleavage of the intein. rHGI was obtained in its native form with no additional residues at either the N- or C-terminus. Yield of rHGI was improved by introducing a trypsin sepharose affinity chromatography step resulting in ~ 670 fold purification. The biochemical characteristics of rHGI point to its close similarity to seed HGI-III not only in its structure but also in its inhibitory characteristics toward bovine trypsin and chymotrypsin. The expression and purification strategy presented here promises to produce BBIs in their natural form for pharmacological and therapeutic use. rHGI with a native N-terminus and no additional residues of purification tags was used as a platform to study 1) the role of the conserved array of seven disulfide bridges in thermal stability and 2) the effect of interactions between two monomers of rHGI that form the dimer. The contributions of two disulfide bonds (C16-C70 and C20-C66) in the trypsin domain to thermal stability and functionality were evaluated using disulfide deletion variants of the 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. Legume seed BBIs that inhibit mammalian proteases associate end to end and exist as dimers in solution. The structural basis for governing dimerization is poorly understood. A three dimensional model of the horsegram HGI-III dimer was computed using the winter pea seed inhibitor (1PBI) structure as a template. A novel aspect of the dimer model is a knob-in-the-hole like interaction between Asp76 and Lys71 at the Cterminus of the inhibitor monomer. It is postulated that the loop created by this interaction enables a very strong interaction between Asp75 of one monomer and Lys24 of the opposite monomer, which leads to dimerization of the molecule. Accordingly, site directed mutagenesis followed by size exclusion chromatography and SDS-PAGE demonstrate that mutation of either Lys71, Asp76 or Asp75 leads to the formation of a monomer, which are kinetically similar but less thermo stable than the dimer. Molecular dynamics simulation of the dimer reveals that the intra molecular interaction is stable throughout the entire simulation. In contrast the intermolecular interaction is initially destabilised due to the side chain orientation of Asp75. It can therefore, be concluded that the stable salt bridge predisposes the geometry of Asp75 for its specific interaction with Lys24 of the opposite monomer. The importance of the C-terminal loop is reinforced by the dimeric nature of the fourth mutant (K71D/D76K), in which only the positions, but not the chemical nature of the interactions between Lys71 and Asp76, are interchanged. The C-terminal loop stabilized by a knob-in-the-hole interaction is the lynchpin, which predisposes the specific interaction between two monomers resulting in the dimerization of the inhibitor. These results underscore the importance of specific electrostatic interactions for legume BBI dimerization. A preliminary model of HGI-III -trypsin interaction indicates that only a binary complex is formed indicating that HGI-III must first dissociate into the monomer. All these result suggest that HGI-III in situ adopts a stable and well packed dimeric state, as a mechanism for controlling its stability to modulate its physiological role as a plant defense protein.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: Bowman-Birk inhibitors, legumes, horsegram, disulfide bridges
Subjects: 500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology
600 Technology > 08 Food technology > 22 Legumes-Pulses
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 20 Mar 2015 10:03
Last Modified: 28 Aug 2018 05:58
URI: http://ir.cftri.com/id/eprint/11768

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