Solution structure of a sweet protein: NMR study of MNEI, a single chain monellin.

The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/(2)H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278-328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT TF (-0.61). The topology of MNEI, like that of natural monellin and of SCM, another single-chain monellin, is typical of the cystatin superfamily: an alpha -helix cradled into the concave side of a five-strand anti-parallel beta -sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main P-strands that surround the helix in the crystal structure or SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 muM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6-9 and 94-96.