DETERMINATION OF THE MOLECULAR-CONFORMATION OF MELANOSTATIN USING C-13,N-15-REDOR NMR-SPECTROSCOPY

Results of a C-13,N-15-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 angstrom to 4.9 angstrom are measured for a series of selectively C-13,N-15-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 phi(Leu), psi(Leu), and phi(Gly) in the tripeptide. Together with conservative energetic considerations, the REDOR-measured distances determine a family of backbone structures for the tripeptide. phi(Leu) is limited to the range -70-degrees to +20-degrees, psi(Leu) from 130-degrees to 145-degrees, and phi(Gly) from -5-degrees to +65-degrees. 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 angstrom (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.