Mechanisms For The N-terminal Domain Of HMW-GS Contributing To The Processing Quality Of Wheat Flour

Wheat glutenin,especially high molecular weight glutenin subunits?HMW-GS?,determines the formation of gluten networks and the processing quality of wheat flour.Thereinto,three domains of HMW-GS exhibite various critical importances in the functions of whole subunits.Therefore,clarifying the relationship between the structural and functional properties of therse domains has become the hot issue urgently need to be addressed in the filed of cereal research.In wheat,1Dx5 is the typical representative of HMW-GS with highest processing quality.Therefore,the N-terminal domain of 1Dx5?1Dx5-N?was chosen as the experimental subject in the present study.We investigated the effects of intermolecular interaction forces on the aggregation behavior of 1Dx5-N,and the functional contributions of 1Dx5-N to the formation of gluten polymers,gluten networks structure and visco-elasticity of dough,etc.Moreover,the disulfide linkages of 1Dx5-N were dissected.The results will facilitate our understanding of the structural and functional properties of the N-terminal domain of HMW-GS,strengthen our theoretical level of manipulating the processing quality of wheat flour and enrich the academic research content of cereal science.?1?The Aggregation Behavior of Recombinant 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 denaturant sodium dodecyl sulfonate?SDS?on 1Dx5-N polymer were investigated by native polyacrylamide gelelectrophoresis?PAGE?,non-reducing/reducing SDS-PAGE,intrinsic fluorescence,size exclusion chromatography,dynamic light scattering and circular dichroism.Results showed that 1Dx5-N formed a soluble aggregate in aqueous solutions,clarifying that the N-terminal domain was not the molecular basis for the insolubility of whole subunit.Meanwhile,the hydrophobic interaction was more potent in promoting the aggregation of 1Dx5-N than disulfide bonds.Moreover,hydrophobic interactions reinforced the formation of disulfide bonds.Edible salts NaCl improved the aggregates formation of 1Dx5-N through disulfide bonds.However,Na₂CO₃ at low concentration??100 mM?dissociated the disulfide bonds,and Na₂CO₃ at relatively high concentration?>200 mM?induced other types of cross-linking between amino acids in 1Dx5-N except disulfide bonds.?2?Heat and edible salts induced aggregation of 1Dx5 and its effects on the interfacial properties.The aggregation of 1Dx5-N induced by heating?37°C,50°C,65°C,80°C?and edible salts?NaCl,Na₂CO₃?was systematically investigated using circular dichroism,dynamic light scattering and size exclusion HPLC as major tools.Results revealed that heating with NaCl induced the formation of macro-molecular weight aggregates of 1Dx5-N?>>670 kDa?through disulfide bond and hydrophobic interactions.Whereas heating with Na₂CO₃ under alkaline pH broken the disulfide bonds and decreased protein surface hydrophobicity,leading to the formation of relatively low-molecular aggregates?44.3-670 kDa?.The macro-molecular weight aggregates negatively impacted protein capacity to reduce surface tension,nevertheless this greatly improved the formation of“gel-like”structure at the air/water surface,which would be beneficial for the gas-retention ability of wheat dough.The relatively low-molecular aggregates efficiently reduced the surface tension of 1Dx5-N,but led to weakened interfacial layer,which could be detrimental for the gas-retention of dough.?3?Role of 1Dx5-N in the functional and structural properties of wheat dough.Effects of N-terminal domain of high molecular weight glutenin subunit?HMW-GS?1Dx5?1Dx5-N?on functional and structural properties of wheat dough were determined by farinographic and rheological analysis,size exclusion chromatography,non-reducing/reducing SDS-PAGE,total free sulfhydryl determination,scanning electron microscopy and Fourier transform infrared spectroscopy.Results showed that1Dx5-N improved the quality of dough with the increased water absorption,dough stability time,elastic and viscous modulus,and the decreased degree of softening,loss tangent.These improvements could be attributed to the formation of the macro-molecular weight aggregates and massive protein networks,which were favored by1Dx5-N through disulfide bonds and hydrophobic interactions.Additionally,1Dx5-N drove the transition of?-helix and random coil conformations to?-sheet and?-turn conformations,further providing the structural basis for the enhancement of dough strength.Moreover,all the positive effects of 1Dx5-N were reinforced by NaCl.?4?Dissecting the disulfide linkage of 1Dx5-N and its contributions to the stability of protein and dough functionality.The N-terminal domain of HMW-GS1Dx5?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 sitedirected 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,Cys40also 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 linkages of HMW-GS through hydrophobic interaction.Additionally,compared to 1Dx5-N and other mutants,only the C25S mutant efficiently improved the quality of dough by enhancing the formation of gluten network structures and disulfide linkage,modifying the secondary structures of gluten,etc.The substitution of Cys25facilitated the participation of Cys10 and Cys40 in the formation of gluten network structure.Based on above results,this study constructed the cross-linking model of Cys residues at different sites in 1Dx5-N and dissected their contributions to dough functionality.Additionally,Na₂CO₃-induced?-elimination of cystine might occur in glutenin without heating.