Structural characterization of β-endorphin through the design, synthesis, and study of model peptides

Analysis of the amino acid sequence of β-endorphin reveals a potential amphiphilic α- or π-helix in the COOH-terminal region, residues 13-29. We have proposed that the biological properties of β-endorphin are determined by the connection of a highly specific opiate receptor recognition sequence at the NH₂ terminus, having the same sequence as (Met⁵)enkephalin, to this amphiphilic helical structure at the COOH terminus, via a hydrophilic peptide link [Taylor et al., J. Am. Chem. Soc. 103:6965 (1981)]. This proposal was investigated by studying peptides 1 and 2, two 31-amino acid analogues of β-endorphin designed to retain the general characteristics of the amphiphilic helix postulated for β-endorphin, but using amino acid sequences with minimal homology to the natural sequence in that region. In addition, peptide 2 has minimal homology to β-endorphin in the proposed hydrophilic linking region, so that peptide 2 residues 6-31 have only four residues homologous to the corresponding residues in the natural sequence. Both peptide 1 and peptide 2 had 50% α-helical structure in 50% aqueous 2,2,2-trifluoro-ethanol, compared with 40% found for β-endorphin, and both model peptides exhibited an even greater resistance to proteolysis in rat brain homogenates than did β-endorphin. Peptide 1 had higher affinities for δ and μ-receptors than did β-endorphin, while retaining the same δ/μ selectivity, and had a greater potency than did β-endorphin in opiate assays on guinea pig ileum. In opiate assays on rat vas deferens, which are highly specific for β-endorphin, peptide 1 had the same activity (IC₅₀ = 61 ± 12 nM) as did β-endorphin (IC₅₀ = 52 ± 3 nM). These results strongly support the importance of an amphiphilic helical structure in β-endorphin residues 13-29 which contributes to the opiate receptor interactions and determines the resistance to proteolysis of the whole molecule. Peptide 2 showed δ-receptor binding and guinea pig ileum activities similar to those of β-endorphin, but behaved quite differently in μ-receptor binding assays (in Tris buffer at 25°C) and displayed a somewhat weaker activity on rat vas deferens (IC₅₀ = 450 ± 15 nM). This suggests that the hydrophilic region of β-endorphin (residues 6-12) plays a role in determining its opiate receptor selectivity.