Uncovering pH-driven metastable transitions and thermal aggregation of HMW-GSs via all-atom molecular dynamics simulations

High-molecular-weight glutenin subunits (HMW-GSs) are key contributors to wheat dough viscoelasticity, with their conformational adaptability playing a vital role in gluten network formation. In this study, we employed a two-stage molecular dynamics simulation approach to investigate how acidic (pH 2) and alkaline (pH 12) conditions modulate the metastable states of three x-type HMW-GSs-Ax1, Bx7, and Dx2-and how these conformations respond to thermal aggregation. Results revealed clear subunit-specific differences in conformational plasticity. Under extreme pH conditions, Ax1 and Bx7 displayed greater RMSD fluctuations and broader radius of gyration distributions compared to Dx2, demonstrating enhanced structural flexibility. Hydrogen bond lifetime analysis indicated that Bx7 maintained longer-lived interactions across metastable states, implying greater internal stabilization. Upon thermal acceleration at 400 K, Ax1 rapidly progressed toward aggregation-prone conformations, whereas Dx2 preserved partial structural integrity, reflecting its superior thermal resilience. Covariance-based cross-correlation analysis further showed extensive domain decoupling in Ax1 and Bx7, in contrast to the more coordinated dynamics of Dx2. By linking metastable ensembles to aggregation behavior, this study provides mechanistic insights into gluten protein adaptability. These molecular insights may also serve as a reference for understanding hydration, gelation, and viscoelasticity of gluten networks in wheat-based food systems.