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Napin from Brassica juncea: Thermodynamic and structural analysis of stability.

Jyothi TC, Sinha S, Singh SA, Surolia A, Appu Rao AG

Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570020, India.

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; DeltaC(P1) and DeltaC(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, Nleft arrow over right arrowIleft arrow over right arrowU with DeltaG(1H2O) and DeltaG(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 A to 35.0 A 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.

Published 2 July 2007 in Biochim Biophys Acta, 1774(7): 907-19.
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