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Low Molecular Weight Proteins of Mustard Seed (Brassica Juncea): Structure, Function and Interaction with Intrinsic Ligands

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Title Low Molecular Weight Proteins of Mustard Seed (Brassica Juncea): Structure, Function and Interaction with Intrinsic Ligands
 
Creator Jyothi, T. C.
 
Subject 29 Protein Chemistry
30 Spices/Condiments
03 Proteins
 
Description Napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits - one small (29 amino acid residues) and one large (86 amino acids residues) - held together by disulfide bonds.
The role of disulfide linkages, electrostatic as well as hydrophobic interactions on napin stability was investigated through spectroscopic methods. The subunits are hydrophilic in nature and possess extended structure. With the addition of 0.5 M NaCl, surface hydrophobicity of napin decreases, while the helical content increases by 25%. In presence of NaCl, emission maximum shifts towards shorter wavelength and the Stern-Volmer constant decreases from 6.5 M-1 to 3.4 M-1, indicating compaction of napin. Na2SO4 has no significant effect on the structure due to lack of hydrophobic core. In presence of monohydric alcohols and trifluoroethanol, there is an increase in ordered structure. The study indicates that the structure of napin, which is hydrophilic in nature, is stabilized by electrostatic interactions, in addition to disulfide linkages.
Thermal unfolding is characterized by a three state transition with TM1 and TM2 at 50.5°C and 62.8°C, respectively; rCP1 and rCP2 are 2.05 kcal mol-1 K-1 and 1.40 kcal mol-1 K-1, respectively. In the temperature range 37 – 45°C, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N D I D U with rG1H2O and rG2H2O values of 5.2 kcal mol-1 and 5.1 kcal mol-1 at 27°C, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from 20 Å to 35.0 Å due to unfolding by guanidinium hydrochloride. In silico alignment of sequences and homology modeling of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin.
The factors responsible for the binding of sinapic acid, phytic acid and allyl isothiocyanate ligands with napin have been studied by spectroscopic methods. Sinapic acid and phytic acid binds to napin with a binding constant of 3.5 ´ 103 and 1.6 ´ 105 M-1 respectively. The available lysine content of napin – allyl isothiocyanate complex decreased significantly from 6 mole/mole to 1.8 mole/mole of napin. Phytic acid and allyl isothiocyanate induces the conformational change in napin structure. Microenvironment of tyrosine in napin changed upon binding to allyl isothiocyanate.
 
Contributor Appu Rao, A.G.
 
Date 2007-05
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Identifier http://ir.cftri.com/1644/1/Jyothi_TC.pdf
Jyothi, T. C. (2007) Low Molecular Weight Proteins of Mustard Seed (Brassica Juncea): Structure, Function and Interaction with Intrinsic Ligands. Doctoral thesis, University of Mysore.