Method for Correlation of Proton Magnetic Resonance Assignments in Different Solvents: Conformational Transition of Oxytocin and Lysine Vasopressin from Dimethylsulfoxide to Water

Abstract

The peptide amide, primary carboxamide, and aromatic proton resonances were assigned to specific hydrogens of oxytocin and lysine vasopressin (Lys-VP) in water at 230 at pH 2.5 and 4.2, respectively. We started with the spectral assignments of oxytocin and Lys-VP determined in deuterated dimethylsulfoxide (Me2SO) and monitored the course of each of these resonances as the proportion of water to Me2SO was gradually increased. Changes in each of the two hormones in chemical shifts and in some coupling constants indicate that conformational alterations occur in both oxytocin and Lys-VP during the solvent transition from Me2SO to water. This study is a specific application of a general method for correlating spectral assignments in different solvents and for monitoring conformational changes accompanying solvent transitions. Application of this technique requires only that the solvent components be miscible over the entire transitional range, that spectral changes of the solute be simple enough to follow, and that the associated structural changes of the solute be "rapid on the proton magnetic resonance time-scale."It is generally assumed that peptide hormones interact with their specific receptors at the surface membrane. The systemic circulation of a peptide hormone takes place in an essentially aqueous system, but the hormone-receptor interaction occurs at a hydrophilic-hydrophobic interphase. It is therefore important to perform conformational analyses of peptide hormones in both aqueous and nonaqueous solvents.Prerequisite to such investigations is the assignment of individual resonances to specific protons. Detailed assignments have been achieved with peptide hormones (1, 2) and antibiotics (3-10) in nonaqueous media, but not in H20. The intense absorption of the H20 resonance interferes with the double resonance experiments necessary for correlating the peptide NH and aCH resonances of individual aminoacid residues in native peptides.In this communication we report a new and convenient method for assignment of amide proton resonances in water. Starting with the proton magnetic resonance (PMR) assignments of oxytocin (1) and lysine-vasopressin (Lys-VP) (2) in dimethylsulfoxide (Me2SO), the chemical shifts of individual resonances are followed through the stepwise transition of the solvent from Me2SO to water. This study is a specific application of a general method for correlating spectral assignments in different solvents, provided that the solvent components are completely miscible over the entire range of the transition, that the concomitant spectral changes can be followed and that the associated structural changes are "rapid on the PMR time scale." MATERIALS AND METHODSOxytocin and Lys-VP (Fig. 1) were prepared by the solid-phase method of peptide synthesis (11), and exhibited 450-500 U/mg of avian vasodepressor (12) and 270-300 U/mg of rat pressor (13) activities, respectively, which is in line with potencies previously described for these hormones (14). Stock solutions of each hormone (3% w/v) w...

Unequivocal assignments of NH proton magnetic resonance bands in oxytocin using 2H‐ and 15N‐substituted molecules

“…This resonance can therefore be assigned unambiguously to the tyrosine NH proton. This is in agreement with the results of both Walter and coworkers [3] and Brewster and Hruby [4]. However, both these groups assigned this resonance essentially by elimination, since the doubleresonance experiment in H20 was inconclusive [4], and the resonance disappeared in DMSO/water mixtures containing between 30% and 80% water [3].…”

“…1 b) shows clearly that the NH resonance at 7.94 ppm in oxytocin is that of isoleucine. This assignment is in agreement with that of Brewster and Hruby [4], but not with that of Walter et al [3]. The second type of isotopic substitution we have employed for assignment purposes is exemplified by 8-[a-2 H-Leu]-oxytocin ( fig.…”

“…Assignment of the NH resonances required the use of H20 solution, and 'blind' irradiation of the aCH resonances underneath the H20 absorption band. The assignments of Brewster and Hruby [4] are in substantial disagreement with those of Walter and coworkers [3].…”

“…Two reports of assignments of the ~ H NMR spectra of oxytocin in aqueous solution have appeared [3,4]. Walter and his colleagues [3] took the assignments made by double resonance experiments in dimethylsulphoxide (DMSO) solution [5], and attempted to transfer them to aqueous solution by measuring…”

“…Walter and his colleagues [3] took the assignments made by double resonance experiments in dimethylsulphoxide (DMSO) solution [5], and attempted to transfer them to aqueous solution by measuring…”