Proton Magnetic Resonance Study of Peptide Conformation: Effect of Trifluoroethanol on Oxytocin and 8-Lysine-Vasopressin

Walter, Roderich; Glickson, Jerry D.

doi:10.1073/pnas.70.4.1199

Cited by 15 publications

(3 citation statements)

References 23 publications

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“…USA 71 (1974) mational effects observed upon alterations of the protonation state of the amino group in oxytocin. Conformational alterations as a result of changes in the solvent composition have been encountered with oxytocin; depending on the nature of the solvent transition, these changes either stabilized or destabilized the proposed a-turn in the 20-membered ring of the hormone (17,18).…”

Section: Resultsmentioning

confidence: 99%

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

Deslauriers¹,

Walter²,

Smith³

1974

Proc. Natl. Acad. Sci. U.S.A.

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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

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Section: Resultsmentioning

confidence: 99%

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

Deslauriers¹,

Walter²,

Smith³

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…In these conditions the amide protons of Tyr 2 , Ile 3 , Leu 8 and Gly 9 all shifted significantly upfield ( δ of 0.7, 0.2, 0.6 and 0.4 ppm, respectively) relative to water (Table 6) indicating a modification of the peptide conformation in changing from water to 7 : 3 TFE : H 2 O. In the spectrum of OT (3), similar upfield shifts of the amide protons of Ile 3 , Leu 8 and Gly 9 have been observed upon the titration of TFE into DMSO [49][50][51].…”

Section: Conformational Analysis By Nmr Spectroscopymentioning

confidence: 74%

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, raman, CD spectroscopy and molecular modeling

2005

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The synthesis and biological evaluation of penicillamine 6 -5-tert-butylproline 7 -oxytocin analogs and comparison with their proline 7 -oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen 1 ,Pen 6 ]-oxytocin (1, pA 2 = 8.22, EC 50 = 6.0 nM) and [dPen 1 ,Pen 6 ,5-tBuPro 7 ]-oxytocin (2, pA 2 = 8.19, EC 50 = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using 1 H NMR, laser Raman and CD techniques. In H 2 O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln 4 and Pen 6 in the major trans-conformer of 1 as well as for Gln 4 in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 ± 0.2 kcal/mol) for isomerization of the amide N -terminal to 5-tBuPro 7 relative to OT was calculated from measuring the coalescence temperature of the Gly 9 backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C β -S-S-C β dihedral angles of +110-115°and ±90°, respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a χ CS−SC dihedral angle value close to 115°was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a ±90°angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a ±90°χ CS−SC dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114°and +116°. Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104°. In sum, the conformation about the disulfide in [dPen 1 ,Pen 6 ]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen 1 ,Pen 6 ,5-tBuPro 7 ]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro 7 (5R)-tert-butyl group, the Leu 8 isopropyl sidechain and the Pen 6 β-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110°may be a prerequisite for their binding at the receptor.

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“…An important aspect of such a conformational analysis is determining which specific hydrogens of oxytocin are accessible to solvent and which are inaccessible as a result of intramolecular interactions. Four methods have been employed to measure solvent exposure of peptides: (1) rates of NH proton replacement by hydrogen isotopes (Hvidt and Nielsen, 1966;Molday et al, 1972;Stern et al, 1968), (2) temperature dependence of chemical shifts of NH resonances (Kopple et al, 1969; Ohnishi and Urry, 1969), (3) dependence of NH chemical shifts on the composition of a suitable solvent mixture (Pitner and Urry, 1972a,b;Kopple and Schamper, 1972a;Walter and Glickson, 1973), and (4) the degree of resonance broadening in the presence of a paramagnetic substance (Kopple and Schamper, 1972b). Each method has limitations.…”

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Proton nuclear magnetic resonance double resonance study of oxytocin in aqueous solution

Rowan¹,

Tp²,

Dadok³

et al. 1976

Biochemistry

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Peptide NH resonances in the 250 MHZ 1H nuclear magnetic resonance (NMR) spectrum of oxytocin in H2O were assigned to specific amino acid residues by the "underwater decoupling" technique (i.e., decoupling from corresponding CalphaH resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster an V. J. Hruby ((1973), Proc. Natl. Acad. Sci. U.S.A. 70, 3806) and A. F. Bradbury et al. ((1974), FEBS Lett. 42, 179). Three methods of assigning NH resonances of peptides--solvent titration, underwater decoupling, and isotopic labeling--are compared. As the solvet composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE's) of 14 +/- 5 were observed for the Tyr ortho CH and meta CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipoledipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE's therefore indicate intimate contact between water and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between water and NH group varies with Ph in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shiedled from the solvent.

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Proton Magnetic Resonance Study of Peptide Conformation: Effect of Trifluoroethanol on Oxytocin and 8-Lysine-Vasopressin

Cited by 15 publications

References 23 publications

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

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

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, raman, CD spectroscopy and molecular modeling

Proton nuclear magnetic resonance double resonance study of oxytocin in aqueous solution

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