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Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydantoins (direct micro-sequence analysis). Evidence for residue 40 (isoleucine) was provided by direct micro-sequence analysis of 2.0 nmol of acetylated CRF selectively cleaved at its arginine residues by trypsin prior to analysis. The thermolytic COOH-terminal fragment isoleucyl-alanineamide was characterized as its dansyl derivative. Based on the analytical data, the following primary structure is proposed for ovine hypothalamic CRF: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp -Ile-Ala-NH2. In agreement with this proposal, the synthetic replicate of CRF is highly potent in stimulating secretion of both corticotropin and beta-endorphin-like immunoactivities.

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

Several neuropeptides injected intracisternally were assessed for their effects on gastric secretion in rats. Bombesin (1 microgram) completely suppressed gastric acid secretion, produced the volume of gastric secretion, and partially blocked insulin- or 2-deoxy-D-glucose-induced stimulation of gastric acid output. The inhibitory effect of this peptide is dose-dependent, long-acting, reversible, and specific. Bombesin response appears to be central nervous system-mediated; its expression is not dependent on the vagus nerve or the adrenal glands, and does not rely on a decrease in gastrin secretion. Among seven other peptides tested, only beta-endorphin and a potent gonadotropin releasing-factor (gonadoliberin) agonist significantly reduced gastric acid secretion, with an activity ca. 100 times less than that of bombesin. The presence of bombesin-like material in rat brain and the high potency of bombesin to inhibit gastric secretion suggest that this peptide may be of physiologic significance as a chemical messenger involved in brain modulation of gastric secretion.

Inhibin, a gonadal protein that preferentially suppresses the secretion of pituitary follicle-stimulating hormone, has been isolated from porcine follicular fluid and characterized as a 32-kDa protein composed of 18-kDa and 14-kDa subunits. In the present work, oligonucleotide probes predicted from amino-terminal inhibin amino acid sequences have been used to isolate, from a porcine ovarian lambda gt11 cDNA library, clones encoding the 18-kDa subunit, or A chain, of inhibin. DNA sequence analysis showed that the inhibin A chain is initially synthesized as a larger precursor protein and is predicted to be a glycopeptide. Inhibin A-chain mRNA is present specifically in the gonads, and its synthesis can be induced by treatment of the animal with gonadotropins. The porcine probe was used to isolate a human inhibin A-subunit cDNA from a placental cDNA library. The human precursor is highly homologous to its porcine counterpart and is predicted to generate an 18-kDa glycosylated inhibin A subunit.

To investigate the contribution of hypothalamic gonadotropin-releasing hormone (GnRH) secretion to the midcycle gonadotropin surge in the human, the response of luteinizing hormone (LH) to competitive GnRH receptor blockade achieved by administration of a range of doses of a pure GnRH antagonist was used to provide a semiquantitative estimate of endogenous GnRH secretion. The LH response to 5, 15, 50, and 150 micrograms/kg s.c. of the NAL-GLU GnRH antagonist ([Ac-D-2Nal1,D-4ClPhe2,-D-Pal3,Arg5,D-4-p-met hoxybenzoyl-2-aminobutyric acid6,D-Ala10]GnRH, where 2Nal is 2-naphthylalanine, 4ClPhe is 4-chlorophenylalanine, and 3Pal is 3-pyridylalanine) was measured in normal women in the early and late follicular phases of the menstrual cycle, at the time of the midcycle LH surge and in the early luteal phase. LH decreased in a dose-response fashion after administration of the GnRH antagonist in all cycle phases (P < 0.0001). When this suppression was expressed as maximum percent inhibition, there was no difference in response during the early and late follicular and early luteal phases. However, at the midcycle surge, there was a leftward shift of the dose-response curve with significantly greater suppression of LH at the lower antagonist doses in comparison to the other cycle phases (P < 0.005), but no difference at the highest dose. Thus, we draw the following conclusions. (i) There is a consistently greater degree of LH inhibition by GnRH antagonism at the midcycle surge at submaximal degrees of GnRH receptor blockade than at other phases of the menstrual cycle in normal women. (ii) This leftward shift of the dose-response relationship to GnRH receptor blockade suggests that the overall amount of GnRH secreted at the midcycle surge is less than at other cycle stages. (iii) These data confirm the importance of pituitary augmentation of the GnRH signal at the time of the midcycle gonadotropin surge in the human.

A polypeptide was purified from rat hypothalamic extracts on the basis of its high intrinsic activity to release corticotropin (ACTH) from cultured rat anterior pituitary cells and its immunoactivity in a radioimmunoassay directed against the NH2 terminus (residues 4-20) of ovine hypothalamic corticotropin-releasing factor (CRF). Based on Edman degradation, peptide mapping, and amino acid analysis, the primary structure of this rat CRF was established to be: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. The hypophysiotropic potency of synthetic rat CRF did not deviate significantly from the potencies of the isolated native peptide or of synthetic ovine CRF. The close structural relationship between rat and ovine hypothalamic CRF is indicated by an 83% sequence homology.

The binding of a radiolabeled bomebesin analogue to rat brain membranes was studied. [125I-Tyr4]Bombesin bound with high affinity (KD = 3 nM) to a single class of non-interacting sites. Binding was specific, saturable (3.8 pmol of sites/g of wet tissue), and reversible. Regional and subcellular distribution studies showed that the density of sites was 7-fold greater in the hippocampus than the medulla/pons and greater in synaptosomal fractions than in mitochondrial or nuclear fractions. The abilities of numerous bombesin analogues to induce hypothermia and to inhibit [125I-Tyr4]bombesin-binding activity correlate well. Numerous amino acid residues near the CONH2-terminal are required for high-affinity binding and biological potency.

Most vertebrate species have more than one form of gonadotropin-releasing hormone (GnRH) in their brains, but it is not clear whether each form has a distinct function. We report that sea bream (Sparus aurata) brains have three forms of GnRH, one of which is described herein and is called sea bream GnRH (sbGnRH). The primary structures of two forms were determined by Edman degradation and mass spectral analysis. The amino acid sequence of sbGnRH is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2. The second peptide is identical to a form originally isolated from chicken brains (cGnRH-II): pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. cGnRH-II is the most ancient form of GnRH identified to date in jawed fish and the most prevalent form throughout the vertebrates. The third form of GnRH has previously been identified as salmon GnRH by cDNA studies and is confirmed here by chromatographic and immunological studies. Phylogenetic distribution of GnRH peptides suggests sbGnRH arose in the perch-like fish as a gene duplication of the existing cGnRH-II or salmon GnRH genes. All three identified GnRH peptides were synthesized and shown to release gonadotropin in vivo in the sea bream. The dominant form of GnRH stored in the pituitary was sbGnRH. Not only was the content of sbGnRH 500-fold greater than that of salmon GnRH but also cGnRH-II was not detected in the pituitary. The latter evidence suggests that sbGnRH is the endogenous releaser of gonadotropin II.

Most of the somatostatin-like activity from pigeon pancreas was found to correspond to small species with an apparent molecular weight of 1500--2500. This species was isolated under conditions minimizing intermolecular interactions and protease activities. The isolated product was characterized by two somatostatin radioimmunoassays, a bioassay, endgroup determination, and amino acid analysis. The structure of the isolated compound was determined to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Additionally, small amounts of des-Ala1-somatostatin, a possible degradation product of pancreatic somatostatin, and a large somatostatin-like species with an apparent molecular weight of 11,000--12,500 were detected. It is concluded that the main somatostatin-like polypeptide isolated from pigeon pancreas is identical to the mammalian hypothalamic tetradecapeptide somatostatin.

Basal levels of immunoreactive (ir) beta-endorphin, corticotropin (ACTH), and prolactin (PRL) in plasma of male rats decrease after dexamethasone pretreatment (400 microgram/kg at 24 hr and 200 microgram/kg at 2 hr before). Inescapable electric footshocks increase ir-beta-endorphin, ACTH, and PRL plasma levels and this effect is blocked by dexamethasone pretreatment. Morphine (20 mg/kg) also increases ir-beta-endorphin, ACTH, and PRL levels. Dexamethasone pretreatment blocks the morphine-induced release of ir-beta-endorphin but does not prevent the morphine-induced release of PRL. Naloxone, the opiate antagonist, decreases basal plasma levels of PRL and partially blocks the stress-induced increase of PRL, but it has no effect on the basal or stress-induced release of ir-beta-endorphin. These results are consistent with the proposal that beta-endorphin may interact with an opiate receptor involved in the regulation of PRL secretion.

A peptide that is recognized by certain antibodies raised against mammalian gonadotropin-releasing hormone has been purified from extracts of salmon brains by gel filtration and high-performance liquid chromatography. The primary structure of this 10-residue peptide is less than Glu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2. This represents a difference of two amino acids between salmon and mammalian gonadotropin-releasing hormone and demonstrates that most of the molecule has been conserved during evolution. The synthetic form of salmon gonadotropin-releasing hormone is less potent than is mammalian gonadotropin-releasing hormone on mammalian cells and is biologically active in salmon.

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (<5-fold) between the D and L nonalkylated betidamino acid-containing Acyline derivatives. Importantly, most betide diastereomers were equipotent with Acyline. In an attempt to correlate structure and observed potency, Ramachandran-type plots were calculated for a series of betidamino acids and their methylated homologs. According to these calculations, betidamino acids have access to a more limited and distinct number of conformational states (including those associated with alpha-helices, beta-sheets, or turn structures), with deeper minima than those observed for natural amino acids.

Corticotropin (ACTH) secretion by the anterior pituitary is stimulated by catecholamines in vivo and in vitro. The nature of the response in vivo is controversial but appears to be mediated by beta-adrenergic receptors, whereas the response is dependent on alpha-adrenergic receptors in cultured anterior pituitary cells. In the present studies, by using a superfusion technique, we demonstrate that catecholamine stimulation of ACTH release from rat anterior pituitaries changes with time from a predominantly beta-adrenergic-mediated event to a predominantly alpha-adrenergic-mediated event. From 0 to 2 hr after initiating the superfusion, release of ACTH from anterior pituitary glands is stimulated up to 2.4-fold by the beta-adrenergic agonist l-isoproterenol. However, the ACTH secretory response to the alpha-adrenergic agonist l-phenylephrine is less than or equal to 5% of that to l-isoproterenol during the same time period. Beginning 2 hr after the start of the superfusion, the responsiveness to the beta-adrenergic agonist declines, and the response to the alpha-adrenergic agonist increases until, 10 hr after removal, greater than 95% of the catecholamine-inducible ACTH release is mediated by an alpha-adrenergic pathway. The addition of interleukin 1 alone to the medium from the beginning of the superfusion does not modify basal ACTH secretion rates and does not affect the acquisition of the response to phenylephrine. However, the presence of interleukin 1 does allow the maintenance of the full ACTH secretory response to isoproterenol. This effect of interleukin 1 is reversed by an interleukin 1 antagonist. These observations suggest an additional way in which immune regulators might interact with the hypothalamic-pituitary-adrenal axis.

In vertebrates, gonadotropin-releasing hormone (GnRH) belongs to a family of decapeptides characterized by the conservation of residues 1, 2, 4, 9, and 10. In the jawed vertebrates only positions 5, 7, and 8 in the GnRH molecules vary. We have now purified two forms of GnRH from the brains of spiny dogfish (Squalus acanthias) by using reverse-phase high-performance liquid chromatography. The primary structures were established by automated Edman degradation and mass spectral analysis. The distinct structure of the first form (dogfish GnRH) is pGlu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-NH2 (pGlu represents pyroglutamyl). The second peptide is identical to a form of GnRH originally isolated from chicken brains (chicken GnRH-II; pGlu-His-Trp-Ser-His-Gly-Trp-Tyr- Pro-Gly-NH2) and is widespread throughout the vertebrates. We are aware of no other species of cartilaginous fish in which the primary structures of two forms of GnRH have been determined. The presence of chicken GnRH-II in dogfish supports the idea that chicken GnRH-II is the oldest GnRH to evolve in jawed vertebrates. With the addition of the dogfish GnRH structure to the family, two main structural branches of GnRH can be delineated. The physiological effects of dogfish GnRH included the release of not only gonadotropin but also growth hormone from goldfish pituitary fragments.

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, Catostomus commersoni--show high (greater than 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 COOH-terminal structural element that is an alpha-helix in corticotropin-releasing factor and urotensin I and a beta-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 S1-nuclease analysis was used to investigate the pattern of inhibin expression in the rat. In a first series of experiments, expression of the alpha, beta A, and beta B subunits of inhibin were monitored in various tissues from male and female rats. Two observations emerge from these studies. First, expression of inhibin subunits was found in gonadal and extragonadal tissues. In addition to the ovary and testis, inhibin alpha, beta A, and beta B RNAs were detected in the placenta, pituitary, adrenal, bone marrow, kidney, spinal cord, and brain. Detection of inhibin RNAs in the brain and spinal cord suggested that these subunits may exert neuroregulatory functions in the central and peripheral nervous systems. Furthermore, the presence of inhibin alpha and beta subunits in the placenta and the pituitary gland, two cell types that have clearly been shown to be regulated by exogenous inhibin, may reflect existing paracrine and/or autocrine processes active in these tissues. The second observation is that expression of inhibin subunit RNAs may vary by severalfold in a tissue-specific fashion. for example, alpha-subunit RNA levels are abundant in the gonads, whereas beta A-subunit RNA is predominant in the placenta and bone marrow. Finally, it is noted that expression of testicular inhibin RNA subunits decreases during sexual maturation. We conclude that the dimers comprised of inhibin subunits possess diverse functions and may act as growth/differentiation factors as well as a hormone.

Synthetic corticotropin (adrenocorticotropic hormone)-releasing factor [CRF; for the sequence, see Vale, W., Spiess, J., Rivier, C. & Rivier, J. (1981) Science 213, 1394-1397] in aqueous solution exists predominantly as a random coil. At concentrations greater than 1 microM, the peptide shows a tendency to self-aggregate with a concurrent slight increase in the apparent alpha-helical content as measured by the CD spectrum. The alpha-helix formed by this molecule is highly amphiphilic--i.e., the hydrophilic and hydrophobic regions are segregated on opposite faces of the helix. As predicted from the potential amphiphilic structure, CRF binds avidly to the surface of single bilayer egg phosphatidylcholine vesicles. This binding appears to obey a simple Langmuir isotherm with the following parameters: Kd = 1.3 +/- 0.6 X 10(-7) M and capacity at saturation (N) = 11.0 +/- 1.0 mmol of peptide per mol of phospholipid. CRF also readily forms an insoluble monolayer at the air-water interface. The monolayer is composed of monomers of the hormone with molecular areas, A'0 = 22 A2 per amino acid, suggesting a compact secondary structure. Judged from the collapse pressure (19.0 +/- 0.1 dyne/cm; 1 dyne = 10 microN) of the monolayer, the amphiphilicity of CRF approximates that of plasma apolipoproteins, a class of proteins of the most pronounced amphiphilic character. These results suggest that the binding of CRF to the cell membrane is accompanied by the induction of an alpha-helical secondary structure and it is this predominantly helical form that is the biologically active form of the peptide.

Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an alpha beta heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the alpha beta heterodimer, the beta beta homodimer stimulates FSH secretion. Luteinizing hormone (LH)-secreting pituitary cells and gonadal androgen-producing cells have long been shown to form a closed-loop feedback axis. Based on recent studies demonstrating the FSH stimulation of inhibin biosynthesis by ovarian granulosa and testis Sertoli cells, an additional closed-loop feedback axis exists between pituitary FSH- and gonadal inhibin-producing cells. Because uncharacterized Sertoli cell factors have been suggested to either stimulate or inhibit androgen production by testicular Leydig cells, we have tested the intragonadal paracrine actions of heterodimers and homodimers of inhibin subunits. In primary cultures of testis cells, the alpha beta heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the beta beta homodimer suppresses androgen production. Furthermore, similar modulatory actions of inhibin-related proteins were found in cultured ovarian theca-interstitial cells and theca explants treated with LH. In contrast, treatment with the inhibin-related proteins alone did not affect gonadal steroidogenesis. Our data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops.

We have prepared 125I-labeled [Tyr4]bombesin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of 125I-labeled [Tyr4]-bombesin was saturable, temperature-dependent, and reversible and reflected interaction of the labeled peptide with a single class of binding sites on the plasma membrane of pancreatic acinar cells. Each acinar cell possessed approximately 5000 binding sites, and binding of the tracer to these sites could be inhibited by [Tyr4]bombesin [concentration for half-maximal effect (Kd), 2 nM], bombesin (Kd, 4 nM), or litorin (Kd, 40 nM) but not by eledoisin, physalemin, somatostatin, carbachol, atropine, secretin, vasocative intestinal peptide, neurotensin, or bovine pancreatic polypeptide. At high concentrations (>0.1 μM), cholecystokinin and caerulein each caused a small (15-20%) reduction in binding of lableled [Tyr4]bombesin. With bombesin, litorin, and [Tyr4]bombesin, there was a close correlation between the relative potency for inhibition of binding of labeled [Tyr4]bombesin and that for stimulation of amylase secretion. For a given peptide, however, a 10-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of amylase secretion, calcium outflux, or cyclic GMP accumulation. These results indicate that dispersed acini from guinea pig pancreas possess a single class of receptors that interact with [Tyr4]bombesin, bombesin, and litorin and that occupation of 25% of these receptors will cause a maximal biological response.

The primary structure of ovine hypothalamic hypophysiotropic luteinizing hormone-releasing factor, LRF, has been established as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2by hydrolysis of the peptide with chymotrypsin or pyrrolidone-carboxylylpeptidase and by analysis of the products by an Edman-dansylation sequencing technique, as well as by mass spectrometry of the derived phenylthiohydantoins. A decapeptide with the proposed primary structure, prepared by total synthesis, gave the same result on sequencing. The synthetic decapeptide possesses the same biological activities as the native ovine LRF. The amino-acid sequence of ovine LRF is identical to that already published for porcine LRF.

Semiempirical energy calculations were performed to determine the conformation of the thyrotropin-releasing factor, L-pyroglutamyl-L-histidyl-L-prolineamide. Our results strongly suggest a “hairpin turn” conformation stabilized by two specific hydrogen bonds. Experimental support for this structure was obtained from proton magnetic resonance by use of chemical shifts and coupling constants with the implied dihedral angles.