Sweeter and Stronger: Structural-Driven Molecular Design to Enhance Sweetness and Stability of the Single Chain Monellin MNEI

Abstract

Sweet proteins are a family of proteins with no structural homology, able to elicit a sweet sensation by interacting with the dimeric T1R2-T1R3 sweet receptor [Picone, D. and Temussi, P.A., 2012, Plant science, 195, 135-142]. Our studies focus on MNEI, a single chain derivative of the natural protein monellin, representing one of the sweetest molecules known to date. MNEI stimulates a strong interest for its biological properties, but is also a well accepted model for protein folding and aggregation studies. In the framework of the “wedge model” [Temussi, P.A., 2009, TIBS, 34, 296-302], we have integrated computational and experimental techniques to enhance sweetness and stability of MNEI against physical and chemical agents [Leone S., et al., 2016, Scientific Reports, 6, 34045]. Based on molecular modeling, NMR [Spadaccini, R., et al., 2016, FEBS Lett, (in press)], and X-ray crystallography, we designed the sweetest protein ever obtained, which combines a sweetness threshold of about 25 nM with a very high and pH-independent thermal stability. Docking studies have provided a rationale basis to explain these properties, hinting at a previously unpredicted role of plasticity in the interaction between this protein and the sweet receptor. Additional aspects of protein aggregation and unfolding properties studied by Molecular Dynamics [Leone S. and Picone, D., 2016, PloS one, 11 (6), e0158372], Atomic Force Microscopy and Optic Spectroscopies will be also discussed.