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

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Relation http://ir.cftri.com/11768/
 
Title Molecular Engineering of the Horsegram (Dolichos
biflorus) Seed Bowman-Birk Inhibitor: Implications of
the disulfide framework on functionality.
 
Creator Vinod, Kumar
 
Subject 03 Biochemistry & Molecular Biology
22 Legumes-Pulses
 
Description 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.
 
Contributor Lalitha, R. Gowda
 
Date 2014
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Identifier http://ir.cftri.com/11768/1/Vinod%20Kumar%20Ph.D.%20Thesis.pdf
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.