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Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichos bif lorus): evidence for ‘hyperreactive’ disulf ide bonds and rate-limiting nature of disulf ide isomerization in folding.

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BBA-2002-2
 
Title Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichos bif lorus): evidence for ‘hyperreactive’ disulf ide bonds and rate-limiting nature of disulf ide isomerization in folding.
 
Creator Rajesh Singh, R.
Appu Rao, A. G.
 
Subject 29 Protein Chemistry
22 Legumes-Pulses
 
Description Horsegram protease inhibitor belongs to the Bowman–Birk class (BBIs) of low molecular weight (8–10 kDa), disulfide-rich, ‘dual’
inhibitors, which can bind and inhibit trypsin and chymotrypsin either independently or simultaneously. They have seven conserved disulfide
bonds. Horsegram BBI exhibits remarkable stability against denaturants like urea, guanidine hydrochloride (GdmCl) and heat, which can be
attributed to these conserved disulfide bonds. On reductive denaturation, horsegram BBI follows the ‘two-state’ mode of unfolding where all
the disulfide bonds are reduced simultaneously resulting in the fully reduced protein without any accumulation of partially reduced
intermediates. Reduction with dithiothreitol (DTT) followed apparent first-order kinetics and the rate constants (kr) indicated that the disulfide
bonds were ‘hyperreactive’ in nature. Oxidative refolding of the fully reduced and denatured inhibitor was possible at very low protein
concentration in the presence of ‘redox’ combination of reduced and oxidized glutathiones. Simultaneous recovery of trypsin and
chymotryptic inhibitory activities indicated the concomitant folding of both the inhibitory subdomains. Folding efficiency decreased in the
absence of the glutathiones and in the presence of denaturants (6 M urea and 4 M GdmCl), indicating the importance of disulfide shuffling
and the formation of noncovalent interactions and secondary structural elements, respectively, for folding efficiency. Folding rate was
significantly improved in the presence of protein disulfide isomerase (PDI). A 3-fold enhancement of rate was observed in the presence of
PDI at molar ratio of 1:20 (PDI/inhibitor), indicating that disulfide bond formation and isomerization to be rate limiting in folding. Peptide
prolyl cis–trans isomerase (PPI) did not affect rate at low concentrations, but at molar ratios of 1:1.5 (PPI/inhibitor), there was 1.4-fold
enhancement of the folding rate, indicating that the prolyl imidic bond isomerizations may be slowing down the folding reaction but were not
rate limiting.
 
Date 2002
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/1267/1/Biochimica_et_Biophysica_Acta_1597_%282002%29_280-291.pdf
Rajesh Singh, R. and Appu Rao, A. G. (2002) Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichos bif lorus): evidence for ‘hyperreactive’ disulf ide bonds and rate-limiting nature of disulf ide isomerization in folding. Biochimica et Biophysica Acta, 1597. pp. 280-291. ISSN 0167-4838