Abstract
Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.
Highlights
Of point mutations affecting the potency of monellin, brazzein and thaumatin[23,24,25]
In order to select the best possible mutations for new MNEI constructs, we analysed the electrostatic surface potentials of MNEI in comparison to a model of its sweeter and well characterised mutant Y65R-MNEI23, which we used as the starting point for the present mutagenesis experiments
C41 is the only cysteine in the sequence and it is not involved in the formation of disulphide bridges, it has been identified as the source of destabilisation of MNEI at extremely high pHs48
Summary
Of point mutations affecting the potency of monellin, brazzein and thaumatin[23,24,25]. More advanced models have been built in terms of topological refinement[25], by taking into account the information deriving from previous mutagenesis studies These models have been able to account for many of the experimental outcomes of mutations of charged surface residues, and proved the possibility to predict at the atomic detail the complexes of mutants of MNEI and brazzein with the sweet receptor. We incorporated in the sequence of the sweetest construct mutation E23Q, which has been recently shown to increase stability at neutral to alkaline pH35 This latter mutation was not expected to affect MNEI sweetness, since the side chain of residue 23 is buried in a hydrophobic pocket and not exposed to interactions with the receptor. The results confirm the predictive value of the wedge model to design MNEI mutations and offer a new picture of the interaction between sweet proteins and the receptor
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