Characterization of an amphiphilic helical structure in β-endorphin through the design, synthesis, and study of model peptides

We have proposed that the biological properties of β-endorphin are determined by the combination of a highly specific opiate-receptor recognition sequence, residues 1-5, at the NH₂terminus and a potential amphiphilic α- or π-helix in the COOH-terminal residues 13-29, connected via a hydrophilic peptide link. In the α-form, the amphiphilic helix has a hydrophobic domain which twists around the helix and covers approximately half of its surface. Other prominent features of the helix are its basicity and the 2 aromatic residues Phe 18 and Tyr 27. Peptide 3 was synthesized and studied to determine how the properties of β-endorphin depend on the general characteristics of the amphiphilic α-helical region. Residues 1-13 of peptide 3 and β-endorphin are homologous. In contrast, residues 14-31 of peptide 3 were chosen to be almost nonhomologous to those or β-endorphin, while having a high helix-forming potential. Indeed, peptide 3 can form an amphiphilic helix in its COOH-terminal region that is similar in the shape of its hydrophobic domain to the helix described for β-endorphin. Peptide 3 had considerable helical structure in aqueous 2,2,2-trifluoroethanol solutions, as does β-endorphin, and had much higher affinities for δ- and μ-opiate receptors than β-endorphin, while it retained the same δ/μ selectivity. Peptide 3 also had a greater resistance to proteolysis in rat brain homogenates than β-endorphin, and a higher opiate activity on the guinea pig ileum. In opiate assays on the rat vas deferens, which are very specific for β-endorphin, peptide 3 retained a high activity (IC₅₀ = 267 ± 48 nM) although it was less active than β-endorphin (IC₅₀ = 41 ± 2 nM). Most important, peptide 3 displayed a potent opiate analgesic effect which lasted considerably longer than that of β-endorphin, when equal doses of the peptides were injected intracerebrally into mice. These results strongly suggest that an amphiphilic helical structure in β-endorphin residues 13-29 contributes to opiate-receptor interactions and determines the resistance to proteolysis of the whole molecule. A comparison of these properties with those of other model peptides shows that the shape of the hydrophobic domain in this helix prevents self-association and nonspecific tissue binding of β-endorphin, that the positive charge in this structure enhances δ- and μ-receptor binding, and that activities on the rat vas deferens and in analgesic assays may have different additional specificities for the aromatic residues on the helix surface.