TERTIARY STRUCTURE OF ENDOTHELIN-1 IN WATER BY H-1-NMR AND MOLECULAR-DYNAMICS STUDIES

The 3D structure in pure water of endothelin-1, a recently discovered potent vasoconstrictor peptide, has been determined at different pH an d temperatures, using two-dimensional H-1 NMR spectroscopy and constra ined molecular dynamics (MD). 170 Inter- and intra-residue NOE interac tions were quantified as volume integrals and translated into distance s. All the coupling constants between the amidic and the alpha-protons have been measured and several dihedral angles, thus obtained, have b een used as constraints for the MD. Some stereospecific assignments ha ve also been performed. A family of eleven structures, satisfying the distance constraints to within 0.3 angstrom, was obtained and showed t hat the C-terminal, determinant for the binding with the receptor, has a well defined conformation. The correlation time measurements gave a n average molecular volume consistent with monomeric species. The tert iary structure at neutral pH is that of a compact molecule, in which t he C-terminal of the peptide folds back toward the alpha-helical segme nt (residues 9-16), in close proximity to the pro-R methyl group of Va l12, as defined by important NOEs involving residues 17-21 and the alp ha-helical core residues 9-14. The results are in agreement with the d euterium exchange experiments, which confirm the existence of a hydrop hobic region also at the site of the C-terminal residues 19-21.