Dissecting the Disulfide Linkage of the N-Terminal Domain of HMW 1Dx5 and Its Contributions to Dough Functionality

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Abstract
The N-terminal domain of HMW-GS 1Dx5 (1Dx5-N) contains three cysteine residues (Cys10, Cys25, Cys40), which are the basis of gluten network formation through disulfide bonds. Disulfide linkage in 1Dx5-N was dissected by site-directed mutagenesis and LC-MS/MS, and its contributions to structural and conformational stability of 1Dx5-N and dough functionality were investigated by circular dichroism, intrinsic fluorescence, surface hydrophobicity determination, size exclusion chromatography, nonreducing/reducing SDS–PAGE, atomic force microscopy, and farinographic analysis. Results showed that Cys10 and Cys40 of 1Dx5-N were the active sites for intermolecular linkage. Meanwhile, Cys40 also exhibited the ability to form intrachain disulfide linkage with Cys25. Moreover, Cys10 and Cys40 played a functionally important role in maintaining the structural and conformational stability and high surface hydrophobicity of the N-terminal domain of HMW-GS, which in turn facilitated the formation of HMW polymers and massive disulfide linkage of HMW-GS through hydrophobic interaction. Additionally, the 1Dx5-N mutants in which Cys were replaced by serine (Ser) presented different effects on dough functionality, while only the C25S mutant produced positive effects compared with wild type 1Dx5-N. Na2CO3-induced β-elimination of cystine might occur in glutenin without heating, which would make it much easier to reduce the nutritional quality of flour products by the cost of lysine. Therefore, these results give a deep understanding of the disulfide linkage of the N-terminal domain of HMW-GS and its functional importance, which will provide a practical guide to effectively generate a superior HMW-GS allele by artificial mutagenesis.
Funding Information
  • China Scholarship Council ([2016]3100)
  • National Natural Science Foundation of China (31130042, 31171630, 31471691)
  • Guangdong Province (2014A010107002, 2015AG10011)
  • Research Institute for Food Nutrition and Health

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