Structural Basis for the Exceptional Thermal Stability of the Boiling-Resistant Sweet Protein MNEI

Monellin, known as the sweetest protein, encounters limitations in the food industry due to its poor thermal stability, prompting modifications to enhance its thermal stability. In our previous work, we utilized Python Rosetta to screen multiple designs, successfully constructing four superstable MNEI (single-chain monellin) mutants that can maintain their sweetness at temperatures up to 100 °C. However, the precise mechanism of increased thermal stability remains unclear. To elucidate the mechanism, we determine the high-resolution crystal structures of four superstable mutants and conduct a comprehensive structural analysis combined with molecular dynamics simulations in our study. Our findings indicate that introduction of mutation sites enhances interactions with surrounding residues in some flexible loop regions, particularly in loop K25-R31, potentially stabilizing flexible regions and may contribute to enhancing the rigidity of the global structure. This study provides a direction for further advancement in mutant modifications.