The soluble recombinant N-terminal domain of HMW 1Dx5 and its aggregation behavior

This study seeks to clarify and determine the fundamental properties of N-terminal domain of high molecular weight glutenin subunits (HMW-GS) 1Dx5 (1Dx5-N). 1Dx5-N was expressed in E. coli and its solubility was measured by spectrophotometry. Effects of edible salts (NaCl, Na₂CO₃), disulfide bond reductant dithiothreitol (DTT) and hydrophobic interactions of denaturant sodium dodecyl sulfonate (SDS) on 1Dx5-N polymer were investigated by native polyacrylamide gelelectrophoresis (PAGE), nonreducing/reducing SDS-PAGE, intrinsic fluorescence, size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD). Results showed that 1Dx5-N formed a soluble aggregate in aqueous solutions by native-PAGE, clarifying the role of N-terminal of HMW-GS in the insolubility of the whole subunits. Meanwhile, the hydrophobic interaction was more potent in promoting the aggregation of 1Dx5-N in aqueous solutions from the results of SEC, DLS and CD. Edible salts, NaCl and Na₂CO₃, could improve the polymer formation of 1Dx5-N through disulfide bonds. Moreover, Na₂CO₃ at high concentrations (>200mM) greatly favored polymer formation by disulfide bonds, and it induced other types of cross-links between amino acids in 1Dx5-N according to nonreducing/reducing SDS-PAGE and fluorescence spectrum. Moreover, the formation of covalent bonds was reinforced by hydrophobic interactions between 1Dx5-N. Therefore, these results provide much novel information on the N-terminal domain of HMW-GS to facilitate the understanding of its functional properties in wheat flour.