Relation of the Conformation of Oxytocin to the Biology of Neurohypophyseal Hormones

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The effects of hydrogen ion concentrations on the carbon-13 nuclear magnetic resonance spectra of oxytocin were investigated. The starting pD of 3.0 was increased stepwise to 8.4. A change of the state of protonation of the N-terminal amino group of oxytocin is accompanied by changes in chemical shifts of carbon-13 nuclei of amino-acid residues located in the 20-membered ring of the hormone. The resonance positions of the acyclic peptide portion, Pro-Leu-Gly-NH2, remain constant. The pD-induced chemical-shift changes of carbons up to five bonds removed from the site of protonation are interpreted in terms of "through-bond" and "through-space" mechanisms. Chemical-shift changes of carbons more than five bonds removed are proposed to have a conformational origin. It is suggested that a change in the charge density of the amino group perturbs the dihedral angle of the -CH2--S-S-CH---moiety of oxytocin, which in turn significantly affects the overall conformation of the 20-membered ring of the hormone.In the course of our studies of the neurohypophyseal hormones oxytociD, arginine-vasotocin, and arginine-and lysine-vasopressin by '3C nuclear magnetic resonance (NMR) we became interested in spectral and conformational changes of the hormones induced by alterations of pH. Changes in the state of protonation of the N-terminal amino group are probably responsible for the changes in chemical shifts of residues located in the 20-membered ring moiety of the hormones. Resonances of the '3C spectra of deamino analogs, derivatives of neurohypophyseal hormones that do not possess an Nterminal amino group, are completely independent of hydrogen ion concentration in the pH range 3-8 (1). The resonance positions of carbons constituting the acyclic C-terminal portion of the neurohypophyseal hormones, and their deamino analogs thus far investigated, remain unaffected between pH 3 and 8.These RESULTSThe '3C spectrum of oxytocin was followed as a function of hydrogen ion concentration (Fig. 1). The starting pD of 3.0 was increased stepwise to 8.4. Smaller increments were chosen in the range between pD 5.8 and 7.1, since the apparent pKa of the terminal amino group of oxytocin is 6.3 (4). As can be seen from Fig. 1

Section: Resultssupporting

confidence: 73%

Long-Range, pH-Dependent Effects on the Carbon-13 Nuclear Magnetic Resonance Spectra of Oxytocin

Deslauriers¹,

Walter²,

Smith³

1974

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“…This position corresponds to position 8 in our model of somatostatin, the position where replacement of L-Trp by D-Trp results in enhanced potency. Our proposed model is also similar in the region of positions 6-11 to the model proposed by Walter et al (21) for the ring portion of oxytocin, except that their model incorporates a type II 3-turn involving residues 2-5 of oxytocin. Here the cystine residue of oxytocin (residues 1 and 6) corresponds to Phe residues 6 and 11 of somatostatin, with the ring size of VI being the same as that in oxytocin.…”

Section: Resultssupporting

confidence: 54%

Conformationally restricted bicyclic analogs of somatostatin.

Veber¹,

Holly²,

Paleveda³

et al. 1978

143

A model for a biologically active conformation of somatostatin is proposed. This model is based primarily on the biological results obtained with novel bicyclic somatostatin analogs having a covalent bridge replacing the side chains of residues 5 and 10, 6 and 11, and 5 and 12, respectively, rather than on physical measurements on the hormone in solution. The high activity of an analog in which Phe6 and Phe11 are replaced by cystine provides evidence that these phenylalanines stabilize the biologically active conformer through "hydrophobic bonding" but do not directly interact with the receptor. The synthesis of the novel bicyclic analogs of somatostatin and the effects of these on the inhibition of secretion of insulin, glucagon, growth hormone, and gastric acid are described.

“…The calculations indicate that there is no preference for hydrogen-bond formation in the cyclic moiety, but that the proposed hydrogen bond of the tail between the Gly peptide NH and the Cys-6 C==O can form. There appear to be two likely positions for the Tyr side chain with respect to the ring; in one it points away from the ring as reported for the preferred conformation of oxytocin in [U-2H](CH3)2-SO, in the other it is folded over the ring as proposed for the biologically active structure of oxytocin (15). Also, two allowed positions were found for the tail, one in which the acyclic moiety is folded over the ring as in the proposed model and another in which it stretched away from the ring.…”

Section: Discussionmentioning

confidence: 96%

Conformational Energy Studies of Oxytocin and Its Cyclic Moiety

Kotelchuck¹,

Scheraga²,

Walter³

1972

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Conformational energies were calculated for oxytocin in water, starting with a conformation proposed from nuclear magnetic resonance measurements in [U-2H] The combined use of conformational energy calculations and nuclear magnetic resonance (NMR) measurements can resolve ambiguities present in both techniques and thereby provide structural information about polypeptides in solution (1, 2).'The main ambiguity in the computational method [encountered in earlier calculations on oxytocin (3) ] is the existence of many local minima in the energy surface of the peptide; a major ambiguity in the NMR method is the inability to obtain a unique structure from the available experimental data. However, if a possible structure is proposed for a polypeptide from NMR data, its energy and those of its variants can be examined to assess the validity of the proposed structure. The resulting conformation is valid only if the proposed structure is close to the correct one, and if reliable geometry, parameters and energy functions are used in the computations. This dual approach is applied here to oxytocin, Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2. (4). Conformational energies were calculated as described in refs. 3 and 5, with united atoms in place of -CH, -CH2, and -CH8 groups. Ring closure of the 20-membered cyclic component of oxytocin was effected at the S-S bond.From NMR data in [U-2H](CH3)2SO (6, 7) and circular dichroism measurements in water (8), a structure ( Fig. 1) has been proposed for oxytocin (9). It consists of two a-turns; the first involves the sequence Tyr-Ile-Gln-Asn in the sixresidue ring, and the second the sequence Cys-Pro-LeuGly-NH2 in the acyclic tail: The hydrogen bond in the first a-turn is between the Asn peptide NH and the Tyr C=0, and that in the second ,-turn is between the Gly peptide NH and the Cys-6 C=0; the orientation of the tail with respect to the ring is maintained by a hydrogen bond between the LUu peptide NH and the Asn side-chain C=0. Among the uncertainties in the proposed structure (9) are: the orientation of the Tyr side chain with respect to the ring, and the Type I or II character (10) of the peptide group between lie and Gin. While the structure of Fig. 1 was based on measurements in [U-2H](CH3)2S0, it is assumed as a starting conformation for determination of the structure in water; variants of this structure were also considered in the computations [which pertain to water at pH 7 (5) ]. We cannot evaluate the validity of this assumption, especially since there is evidence of some difference in the structures of oxytocin and the related lysine vasopressin when dissolved in [U-2H](CH3)2S0 or in H20 (11,12).We first examined the six-residue ring of oxytocin, the cyclic moiety; then we added the three-residue tail, the acyclic moiety, in order to treat the complete molecule. The isolated cyclic moiety considered here is part of the complete oxytocin molecule, and not the molecule tocinamide. I To whom requests for reprints should be addressed.