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

Abstract

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.

Amide hydrogen exchange rates of peptides in H2O solution by 1H nuclear magnetic resonance transfer of solvent saturation method. Conformations of oxytocin and lysine vasopressin in aqueous solution

“…Previous NH exchange studies of these molecules employed the isotope exchange method in D20/dimethyl sulfoxide solvent mixtures (40,41). The current investigation serves as a basis for further extending conformational analyses of these hormones to aqueous media by 'H NMR (34,37,39,(42)(43)(44)(45)(46)(47). The exchange rates of primary amide groups proved especially amenable to measurement by the solvent saturation method.…”

Complete NMR analysis of oxytocin in phosphate buffer

“…[4,6] Furthermore, it has been reported that a conformational difference between DMSO and water greatly affected the amide NH protons. [7] Although NMR spectra of OXT in water have been partially analyzed, there are no reports on the complete 1 H, 13 C, and 15 N resonance assignments because of the difficulty in obtaining NMR spectra sufficient for precise elucidation of the conformation of OXT. [7,8] A phase-shift presaturation technique for water suppression by use of a NOESY pulse sequence is effective for the measurement of NMR spectra in H 2 O.…”

Conformations of a model cyclic hexapeptide, CYIQNC:1H-NMR and molecular dynamics studies

“…While extending the past conformational studies (Glickson, Urry, & Walter, 1972;Johnson, Schwartz, & Walter, 1969;Urry & Walter, 1971) on the OT, including those studies on the cyclic moiety of OT (Kotelchuck, Scheraga, & Walter, 1972;Urry, Ohnishi, & Walter, 1970), Walter, Prasad, Deslauriers, and Smith (1973) studied this hormone and its analogs using a 13 C NMR spectroscopy and reported that OT populated different conformations. Various proposed models for threedimensional 3D structure of OT have been reported to reveal the conformations of the OT that consist of two β-turns; one in the cyclic moiety (YIQNC segment) and the other in the C-terminal sequence of CPLG (Brewster, Hruby, Glasel, & Tonelli, 1973;Cowburn, Live, Fischman, & Agosta, 1983;Gibson & Scheraga, 1967;Glickson et al, 1976;Manning, Coy, & Sawyer, 1977;Urry & Walter, 1971;Walter, 1977). Similar results were confirmed from the conformational studies of OT in D 2 O and DMSO using NMR and molecular dynamics methods (Bhaskaran, Chuang, & Yu, 1992), and obtained the conformation similar to that of the crystal structure of deamino-oxytocin (Hruby, 1987;Wood et al, 1986).…”

“…Because of the difficulty in obtaining NMR spectra sufficient for precise elucidation of the conformations of OT in water (Feeney, Roberts, Rockey, & Burgen, 1971;Glickson et al, 1976), the conformational studies of OT by NMR have been shown to be readily performed in DMSO (Ballardin et al, 1978;Bhaskaran et al, 1992;Kato et al, 1993;Wittelsberger, Patiny, Slaninova, Barberis, & Mutter, 2005) and revealed that the assignment of almost all of the 1 H and 13 C resonances of OT can be obtained in DMSO solvent (Budesínsky et al, 2005;Kato et al, 1993;Ohno, Kawasaki, Okuda, & Yamaguchi, 2010) . So, the study of the cyclic part of OT in DMSO solution rather than in water is desirable.…”