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; 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, N <_-_-> I <_-_-> U 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.
Jyothi
T C
TC
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570020, India.
Sinha
Sharmistha
S
Singh
Sridevi A
SA
Surolia
A
A
Appu Rao
A G
AG
D055516:2S Albumins, Plant; D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002845:Chromatography; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D055516:2S Albumins, Plant; D010940:Plant Proteins; C050363:napin protein, Brassica napus; D019791:Guanidine