MilliporeSigma
  • Home
  • Search Results
  • A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor.

A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor.

Archives of biochemistry and biophysics (2014-12-21)
Vinod Kumar, Saravanan Murugeson, Neha Vithani, Balaji Prakash, Lalitha R Gowda
ABSTRACT

Legume Bowman-Birk inhibitors (BBIs) that inhibit mammalian proteases exist as dimers in solution. The structural basis governing dimerization of HGI-III (horsegram seed BBI) was investigated. An intra-monomer salt bridge (D76-K71) stabilizes an atypical hook-like conformation at the C-terminus. We postulate that this hook, positions D75 to enable an inter-monomer salt-bridge D75(a)-K24(b), which results in dimerization. We verify this by K71A and D76A mutations of HGI-III. The mutants were both monomers, likely due to destabilization of the C-terminal hook. Dimerization was sustained in a double mutant K71D/D76K that was anticipated to form a similar hook critical for dimerization. Conversely, K24(b) that interacts with D75(a) of the loop is the specificity determining residue that interacts with trypsin to inhibit its activity. The inter-monomer salt bridge D75(a)-K24(b) must be disrupted for the inhibition of trypsin, requiring HGI-III to transition into a monomer. Size exclusion studies and a model of HGI-III-trypsin complex support this notion. Interestingly, isoforms of the inhibitor present in germinated seeds (HGGIs) are monomers; and most strikingly, the C-termini of these inhibitors are truncated with the loss the C-terminal hook critical for dimerization. The tendency of HGI-III to self-associate seems to relate to its physiological function of a storage protein.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
o-Dianisidine dihydrochloride, tablet, 10 mg substrate per tablet
Sigma-Aldrich
o-Dianisidine dihydrochloride, ≥95%
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
Sigma-Aldrich
o-Dianisidine dihydrochloride, Suitable for use in glucose determination
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
Sigma-Aldrich
Acetonitrile solution, contains 10.0% acetone, 40.0% 2-propanol, 0.05% formic acid
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (w/v) ammonium formate, 0.1 % (v/v) formic acid, 5 % (v/v) water, suitable for HPLC
Sigma-Aldrich
Acetonitrile solution, contains 0.035 % (v/v) trifluoroacetic acid, suitable for HPLC
Supelco
Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
N-p-Tosyl-L-phenylalanine chloromethyl ketone, ≥97% (TLC), powder
Sigma-Aldrich
Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride, ≥98%
Sigma-Aldrich
Isopropyl β-D-1-thiogalactopyranoside, ≥99% (TLC)
Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
Acetonitrile, ≥99.5%, ACS reagent
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG
Sigma-Aldrich
Acetyl chloride, reagent grade, 98%
Supelco
Acetonitrile, HPLC grade, ≥99.93%
Sigma-Aldrich
DL-Phenylalanine, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Supelco
Acetonitrile, analytical standard
Sigma-Aldrich
Acetyl chloride, puriss. p.a., ≥99.0% (T)
Sigma-Aldrich
Formic acid solution, BioUltra, 1.0 M in H2O
Sigma-Aldrich
Acetonitrile, ACS reagent, ≥99.5%
Sigma-Aldrich
Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile, biotech. grade, ≥99.93%
Sigma-Aldrich
Formic acid, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥98%
Sigma-Aldrich
Formic acid, ACS reagent, ≥96%
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%