The single-chain monellin (MNEI) displays same sweet potency as the natural monellin protein. To identify critical residues determining its sweetness, residues located at the loops region were selected for mutagenesis analysis. Mutations of positive-charge residues R31, R53, and R82 consistently led to obvious decrease of sweetness, whereas mutations of negative-charge residues resulted in variable sweet potency. Of note, the E50N mutant in the loop region linking the 2 natural chains showed significantly increased sweetness. Mutations of this residue to M or K led to similar effects, in accordance with the so-called wedge model for explanation of the sweet protein–receptor interaction. Homology modeling was carried out with the firstly reported crystal structure of sweet taste receptor (from medaka fish) as the template, and molecular docking and dynamics simulations suggested that flexible conformations of specific residues located in the loops region play essential roles for the interaction with the receptor and the sweetness of the protein. Moreover, obvious additive effects were found for the sweetness as 2 double-site mutants (E50N/Y65R and E2N/E50N) displayed increased sweetness than their single-site mutants. Our results revealed the flexible loop L23 linking the 2 natural chains as a novel sweetness determinant site of the sweet protein monellin and raised a series of new sweeter mutants, which could provide helpful guidance for molecular designing the sweet-tasting proteins.

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