Napin from Brassica juncea: Thermodynamic and structural analysis of stability

The 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 thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with $T_{M1}$ and $T_{M2}$ at 323.5 K and 335.8 K, respectively; $\Delta C_{P1}$ and $\Delta C_{P2}$ are $2.05 kcal mol^{-1} K^{-1}$ and $1.40 kcal mol^{−1} K^{−1}$, respectively. In the temperature range 310–318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N⇆I⇆U with $\Delta G_{1H2O}$ and $\Delta G_{2H2O}$ values of $5.2 kcal mol^{−1}$ and $5.1 kcal mol^{−1}$ at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from $20 \AA$ to $35.0 \AA$ due to unfolding by guanidinium hydrochloride. In silico alignment of sequences 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. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition