Determination of the Molecular Conformation of Melanostatin Using ¹³C,¹⁵N-REDOR NMR Spectroscopy

Results of a ¹³C,¹⁵N-rotational-echo double-resonance (REDOR) NMR study of the neurohormonal tripeptide melanostatin (Pro-Leu-Gly-NH₂) are reported. The REDOR experiment permits through-space carbon-nitrogen distances to be accurately measured in solid samples. REDOR distances spanning the range 2.4 Å to 4.9 Å are measured for a series of selectively ¹³C,¹⁵N-enriched melanostatins and are in good agreement with those reported in the X-ray structure. The REDOR distances provide effective constraints on the values of the dihedral angles ϕ_Leu, ψ_Leu and ϕ_Gly in the tripeptide. Together with conservative energetic considerations, the REDOR-measured distances determine a family of backbone structures for the tripeptide. ϕ_Lue is limited to the range −70° to +20°,ψ_Leu from 130° to 145°, and ϕ_Gly from −5° to +65°. For a 14-atom backbone fragment, the pairwise, minimum root-mean-square deviation (RMSD) between the family of REDOR structures and the X-ray conformation is 0.437 ± 0.224 Å (avg ± std dev). Based upon this work, a general strategy for selecting labeling sites in peptides and proteins to determine their backbone conformations is described.