Reductive unfolding and oxidative refolding of a Bowman-Birk inhibitor from horsegram seeds (Dolichos biflorus): evidence for "hyperreactive" disulfide bonds and rate-limiting nature of disulfide isomerization in folding.
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 (k(r)) 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.
Singh
R Rajesh
RR
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570013, India.
Appu Rao
A G
AG
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol; D007887:Fabaceae / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013439:Sulfhydryl Reagents; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry / Q000235:genetics; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D013439:Sulfhydryl Reagents; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors; D004229:Dithiothreitol