Abstract
The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible epsilon-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC(50): 100-300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC(50): 0.6-50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.
Highlights
EXPERIMENTAL PROCEDURESPreparation of Peptides—Peptides were synthesized automatically (MilliGen 9050 peptide synthesizer) by the solid-phase method using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry in the continuous flow mode as described previously for the synthesis of corticotropin releasing factor (CRF) analogs [20]
The biologically important peptide hormones corticotropin releasing factor (CRF)1, glucagon, secretin, vasoactive intestinal polypeptide, growth hormone releasing factor (GRF), calcitonin, parathyroid hormone (PTH), calcitonin gene-related peptide (CGRP), etc. have significant features in common
The structural requirements of linear, long-chain peptide ligands for receptor interaction are more poorly understood than those of small molecules because of their numerous potential receptor interaction sites and great conformational freedom, which disfavors a stable conformation in water, a general obstacle to the determination of their biologically relevant conformation
Summary
Preparation of Peptides—Peptides were synthesized automatically (MilliGen 9050 peptide synthesizer) by the solid-phase method using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry in the continuous flow mode as described previously for the synthesis of CRF analogs [20]. Purification was carried out by preparative HPLC to give final products of Ͼ95% purity by reverse phase-HPLC analysis. The peptides were characterized by mass spectrometry, which gave the.
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