Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable α-helix spanning residues Phe11- Ile26 and an antiparallel β-sheet formed by residues 2-5, 36-38, 41-47, 54- 64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding β-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands β2 and β3 are connected by a small bulge comprising residues Ile38- Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures (<SA>(k)) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 Å for secondary structural regions and 0.70 Å for backbone atoms excluding two loop regions. The average restraint energy- minimized (REM) structure exhibited root-mean-square deviations of 1.19 Å for backbone atoms and 0.85 Å for backbone atoms excluding two loop regions with respect to 20 <SA>(k) structures. The solution structure of SCM revealed that the long α-helix was folded into the concave side of a six-stranded antiparallel β-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.