Structural requirements for binding to the δ opioid receptor: Alkyl replacements at the third residue of deltorphin I

The naturally occurring heptapeptide deltorphin I (Tyr- d -Ala-Phe-Asp-Val-Val-Gly-NH 2 ) exhibits extremely high affinity and selectivity for the δ opioid receptor. In an ongoing investigation of the features of this compound that confer these properties, seven new analogs of the peptide, in which phenylalanine at position three was replaced with amino acids containing alkyl side chains, were synthesized and tested for binding to μ, δ, and κ opioid receptors. These substitutions, including tert -leucine, tert -butylalanine, α-aminobutyric acid, norvaline, norleucine, β-cyclopentylalanine and octahydroindole-2-carboxylic acid, assessed the importance of aromaticity and lipophilicity/steric distribution of the side chain at this position in the binding interaction. Findings indicated that: (i) aromaticity at position three is not required for binding, and (ii) hydrophobic character, size, steric distribution and conformational flexibility influence affinity at the δ receptor. The data suggest that substitutions at the β-carbon of this residue disrupt the binding conformation of the peptide and possibly provide adverse steric effects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43173/1/10989_2004_Article_BF00142239.pd

Opioid receptor affinity and selectivity effects of second residue and carboxy terminal residue variation in a cyclic disulfide-containing opioid tetrapeptide

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74202/1/j.1399-3011.1991.tb00274.x.pd

Biochemical determinants of the IGFBP‐3–hyaluronan interaction

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide\u27s ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA

Using small peptide segments of amyloid-β and humanin to examine their physical interactions

Background: Amyloid fibrils in Alzheimer’s disease are composed of amyloid-β (Aβ) peptides of variant lengths. Humanin (HN), a 24 amino acid residue neuroprotective peptide, is known to interact with the predominant Aβ isoform in the brain, Aβ (1-40). Methods: Here, we constructed smaller segments of Aβ and HN and identified residues in HN important for both HN-HN and HN-Aβ interactions. Peptides corresponding to amino acid residues 5-15 of HN, HN (5-15), HN (5-15, L11S), where Leu11 was replaced with Ser, and residues 17-28 of Aβ, Aβ (17-28), were synthesized and tested for their ability to block formation of the complex between HN and Aβ (1-40). Results: Co-immunoprecipitation and binding kinetics showed that HN (5-15) was more efficient at blocking the complex between HN and Aβ (1-40) than either HN (5-15, L11S) or Aβ (17-28). Binding kinetics of these smaller peptides with either full-length HN or Aβ (1-40) showed that HN (5-15) was able to bind either Aβ (1-40) or HN more efficiently than HN (5-15, L11S) or Aβ (17-28). Compared to full-length HN, however, HN (5-15) bound Aβ (1-40) with a weaker affinity suggesting that while HN (5-15) binds Aβ, other residues in the full length HN peptide are necessary for maximum interactions. Conclusion: L11 was more important for interactions with Aβ (1-40) than with HN. Aβ (17-28) was relatively ineffective at binding to either Aβ (1-40) or HN. Moreover, HN, and the smaller HN (5-15), HN (5-15 L11S), and Aβ (17-28) peptides, had different effects on regulating Aβ (1-40) aggregation kinetics

Characterizing the binding of dopamine D1–D2 receptors in vitro

Dysfunction of the dopaminergic pathway is linked to numerous diseases of the nervous system. The D1–D2 receptor heteromer is known to play a role in certain neuropsychiatric disorders, such as depression. Here, we synthesized an eight amino acid residue peptide, EAARRAQE, derived from the third intracellular loop of the D2 receptor and show that the peptide binds to the D1 receptor with comparable efficiency as that of the full-length D2 receptor protein. Moreover, immunoprecipitation studies show the existence of a heteromeric complex formed both in vitro and in total protein derived from temporal and frontal lobe tissue from normal and depressed subjects. The efficiency of the peptide to block the D1–D2 heteromeric complex was comparable in all the samples tested

Characterizing the binding of dopamine D1–D2 receptors in vitro and in temporal and frontal lobe tissue total protein

Dysfunction of the dopaminergic pathway is linked to numerous diseases of the nervous system. The D1–D2 receptor heteromer is known to play a role in certain neuropsychiatric disorders, such as depression. Here, we synthesized an eight amino acid residue peptide, EAARRAQE, derived from the third intracellular loop of the D2 receptor and show that the peptide binds to the D1 receptor with comparable efficiency as that of the full-length D2 receptor protein. Moreover, immunoprecipitation studies show the existence of a heteromeric complex formed both in vitro and in total protein derived from temporal and frontal lobe tissue from normal and depressed subjects. The efficiency of the peptide to block the D1–D2 heteromeric complex was comparable in all the samples tested