Solution structure, backbone dynamics, and stability of a double mutant single-chain monellin - Structural origin of sweetness

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has been characterized as being as sweet as native two-chain monellin, Dat a from gelfiltration high performance liquid chromatography and NMR has pro ven that SCM exists as a monomer in aqueous solution. In order to determine the structural origin of the taste of sweetness, we engineered several mut ant SCM proteins by mutating Glu(2), Asp(7), and Arg(39) residues, which ar e responsible for sweetness. In this study, we present the solution structu re, backbone dynamics, and stability of mutant SCM proteins using circular dichroism, fluorescence, and NMR spectroscopy. Based on the NMR data, a sta ble alpha -helix and five-stranded antiparallel beta -sheet were identified for double mutant SCM, Strands beta1 and beta2 are connected by a small bu lge, and the disruption of the first beta -strand were observed with SCMDR comprising residues of IIe(38)-Cys(41). The dynamical and folding character istics from circular dichroism, fluorescence, and backbone dynamics studies revealed that both wild type and mutant proteins showed distinct dynamical as well as stability differences, suggesting the important role of mutated residues in the sweet taste of SCM. Our results will provide an insight in to the structural origin of sweet taste as well as the mutational effect in the stability of the engineered sweet protein SCM.