Structural determinants of walnut glutelin solubility for enhancing food industry applications

Walnut glutelin, a major storage protein with inherent low solubility, presents significant challenges for food processing and nutritional applications. To address this limitation, we conducted in silico saturation mutagenesis across all 356 amino acid positions, generating 6764 unique single-point mutations to identify strategies for rational solubility enhancement. Solubility results revealed that the vast majority of mutations (97%) had minimal impact on solubility, with only 1% demonstrating beneficial effects. Stability analysis identified a complex trade-off between solubility and structural stability, where 82% of stabilizing mutations decreased solubility. Remarkably, only 75 mutations (<1% of all tested) simultaneously improved both properties, highlighting the strict constraints for dual optimization. Amino acid composition analysis revealed that wild-type glutelin's abundance of serine residues, characteristic of storage proteins prone to aggregation, could be strategically replaced with charged residues, particularly lysine, to achieve optimal solubility enhancement. Structural analysis demonstrated that beneficial mutations predominantly occurred in surface-exposed regions, especially in flexible loops and turns, while buried regions were unsuitable targets due to structural disruption. Chemical classification analysis confirmed that charge-introducing mutations, particularly targeting aromatic and phosphorylatable residues, represented the most effective strategy for solubility improvement. The convergence of sequence-based and structure-based approaches validated key engineering principles: surface charge introduction, preservation of structural integrity through selective targeting of flexible regions, and avoidance of buried residue modifications. These findings provide a rational framework for designing solubility-enhanced walnut glutelin variants while maintaining structural stability, offering practical insights for improving plant protein functionality in food applications.