Structural homology of corticotropin-releasing factor, sauvagine, and urotensin I: circular dichroism and prediction studies.

Abstract

Three recently isolated peptides, whose sequences have been determined-the corticotropin (adrenocorticotropic hormone)-releasing factor of ovine origin, sauvagine, from the skin of the frog Phyllomedusa sauvagei, and urotensin I from the teleost fish, Catostomu commersoni-show high (>50%) sequence homology. CD spectra of the three peptides in trifluoroethanol indicate predominantly helical character for these peptides. Analysis of the secondary structures by the Chou-Fasman method predicts that the overall structural organization of the peptides is the same. All three possess a long internal helix, spanning about 25 residues, connected by a turn region to a COOHterminal structural element that is an a-helix in corticotropin-releasing factor and urotensin I and a fl-sheet in sauvagine. The values for helical content estimated from the prediction method agree reasonably well with those computed from the CD spectra. This agreement as well as the CD spectra of corticotropin-releasing factor fragment 5-33 support the specific assignments of helical regions derived from the Chou-Fasman analysis. The three peptides exhibit significantly less helical structure in water than in trifluoroethanol as indicated by CD spectra. Hydrophilicity profiles provided comparison of the three peptides in terms of their overall hydrophilicity and the location of the regions of maximal hydrophilicity. A unique distribution of hydrophilic and hydrophobic residues within the internal helices is revealed by helical wheel analysis. Patches of both types of residues are formed following a heptad (four/three) rule. Since the two patches are shifted by one residue relative to one another, together they occupy only one face of the helical surface, a feature distinct from other amphiphilic structures.The presence of a corticotropin (adrenocorticotropic hormone)-releasing factor (CRF) in the hypothalamus was established more than 25 years ago (1-3). Its characterization from ovine hypothalami was achieved only very recently (4, 5). Synthetic CRF has been shown to release ACTH and 1-endorphin both in vitro and in vivo and to cause hypotensive and behavioral effects in rats (4, 6). Sauvagine, a 40-amino acid peptide, highly homologous with CRF, was isolated from the skin of the frog Phylonmedusa sauvagei (7) and displays, among other activities, a number of CRF-like effects, such as release of ACTH and , 1 endorphin (4, 8). The recently isolated peptides in this group, urotensin I from two species of teleost fisht, share significant homology with and possess similar biological activities to CRF and sauvagine (9).CRF, sauvagine, and urotensin I belong to a novel family of peptides from widely differing sources. Their high sequence homology is an indication of a possible ancestral relationship. The fact that they are equipotent in some assays indicates that functionally important parts of the molecule were highly conserved during evolution. It is therefore important to establish the secondary and tertiary structures of the three peptides to a...

The binding protein of corticotropin-releasing factor: Ligand-binding site and subunit structure

“…A mass of M obs ϭ 2,845.1 (Fig. 4a) was assigned to rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) labeled by [Bp 6,32 ]h͞ rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33)) (M calc ϭ 2,845.24; ⌬M ϭ 50 ppm). A mass of M obs ϭ 1,171.3 (Fig.…”

“…In view of the composition of the CRFBP-CRF complex, we concluded from our data that one molecule of h͞rCRF was 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33) and its site of labeling, rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), are shown in green. The N-terminal photophore-containing fragment [Bp 6,32 ]h͞rCRF 6 -33 (6 -8) and its site of labeling, rCRFBP(34 -38), are shown in red.…”

“…For further analysis of the composition of the dimeric species, chemical crosslinking was carried out in the presence of the photoprobe [Bp 1 ]h͞rCRF 1-41 without and with prior irradiation. The crosslinked species were visualized by immunodetection of the photoprobe, using polyclonal antibodies directed against h͞rCRF [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] (anti-h͞rCRF). In agreement with the photoprobe's high affinity to rCRFBP (IC 50 ϭ 0.67 nM; 95% CI, 0.40-0.93 nM), a 42-kDa species that corresponded to [Bp 1 ]h͞ rCRF 1-41 chemically cross-linked to rCRFBP was detected after treatment with sulfo-DST (Fig.…”

“…Therefore, the CID mass spectra of the photoadduct fragment and of synthetic rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) were compared with respect to their low-mass ion regions-as described above (data not shown). The lack of the low-mass ion signals at m͞z ϭ 100, 112, and 129 indicated the labeling of the side chain of Arg-23 by [Bp 6,32 ]h͞ rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33). In agreement with the presence of Pro-13 and Asn-20,24 in rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), the low-mass ion signals at m͞z ϭ 70 and 87 (25) were not absent, but their intensities were significantly decreased.…”

A single amino acid serves as an affinity switch between the receptor and the binding protein of corticotropin-releasing factor: Implications for the design of agonists and antagonists

“…By using CD (28) and NMR (29, 30) spectroscopic methods, evidence has been provided that CRF forms an amphiphilic helix whose hydrophobic patch binds to hydrophobic surfaces (31). On the basis of the helical wheel diagrams for the central parts of the peptides Svg (28) and h͞rCRF (Fig.…”