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.