Nuclear Magnetic Resonance Spectrum of Lysine-Vasopressin and Its Structural Implications

Abstract

The NMR spectra at 220 MHz of iysinevasopressin and its precursors were measured in dimethyl sulfoxide, and the peaks were assigned to specific protons. Information about hydrogen bonding was obtained from the temperature coefficients of the chemical shifts. With these data, and with several chemical-shift positions and coupling constants, structural information was derived for this polypeptide hormone.High-resolution nuclear magnetic resonance (NMR) studies offer the possibility of determining the conformations of macromolecules in solution (1). This method is applied here to the well-known octapeptide hormone lysine-vasopressin (LysVP) in deuterated dimethyl sulfoxide [2H]6Me2SO solution. The interpretation of the NMR spectrum of this polypeptide was aided materially by the availability of the blocked precursors (dipeptide to nonapeptide) used in the synthesis of LysVP, i.e., the changes in the spectrum in the progression from dipeptide to nonapeptide to LysVP provided information on the chemical shift of specific protons, as well as on changes in conformation as each amino acid residue is added.LysVP (I) has the following structure, in which the numbers indicate the positions of the individual amino acid residues:Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2. The half-cys-1 2 3 4 5 6 7 8 9 tine residues in positions 1 and 6 are referred to as Cys-1 and Cys-6, respectively. MATERIALS AND METHODSLysVP was a purified synthetic preparation (2, 3) that possessed about 250 U/mg of rat pressor activity.t The following protected peptide intermediates were also used in this study:Abbreviations for amino acid residues and protecting groups are in accordance with the IUPAC-IUB Tentative Rules [J. Biol. Chem., 24, 2491Chem., 24, (1966 The proton NMR spectra were recorded on a Varian HR-220 spectrometer, and on a Varian HA-100 spectrometer using an internal lock and frequency sweep mode. The sample concentrations were 4-10 g/100 ml in (100 mol %) [2H]6Me2SO, that had been stored over molecular sieves. When the polypeptide concentration was varied in this range, no change in the spectra were detected. All chemical shifts are downfield from the internal standard tetramethylsilane. The temperatuie of the sample was controlled to ±2°C. Homonuclear spin-decoupling on the HR-220 spectrometer was done by the field sweep method, using a General Radio Co. 1107-A interpolation oscillator, and on the HA-100 by the frequency sweep method. The spectra were calibrated by the side-band technique.After the NMR measurements were completed, the LysVP solutions were bioassayed. No appreciable inactivation of the LysVP was detected. RESULTS AND DISCUSSION Assignment of peaksThe peaks of the NMR spectrum of LysVP were assigned by making use of the spectra of the blocked precursors, from the di-to the nonapeptide, together with information on the spectra of other oligopeptides and of the isolated amino acids. The latter could not be used by themselves because of the influence of charges on the a-NH3+ and a-COO-groups. However, an amino aci...

Conformational Studies on Tocinamide and Deaminotocinamide by 220 MHz Nuclear Magnetic Resonance Spectroscopy

“…Clearly, the 20-membered ring compounds by themselves have conformations quite different from that proposed (6,16) for the parent hormone and deamino analog. Provided that solvent effects prove unimportant, this result would suggest the formation of a different kind of ring structure for oxytocin and deamino-oxytocin.…”

“…The use of nuclear magnetic resonance (NMR) to study the structural features of cyclic polypeptides is well established (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Information concerning various conformational features, including peptide backbone structure, rigidity, and presence of intramolecular hydrogen-bonding is obtained by this method.…”

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

“…It has been reported that the Tyr NH absorption in spectra of oxytocin and Lys-VP broadens when they are recorded in [U-2H]Me2SO (1,2,16 (Fig. 3) (Fig.…”

Conformational Studies of Oxytocin, Lysine Vasopressin, Arginine Vasopressin, and Arginine Vasotocin by Carbon-13 Nuclear Magnetic Resonance Spectroscopy

“…Investigation of neurohypophyseal hormones by '3C NMR spectroscopy promises, therefore, to yield valuable information in extending past conformational studies of these peptides by thin-film dialysis (39), chromatographic methods (40), circular dichroism (41), minimization energy calculation (42), and proton NMR (1)(2)(3)(4)(5)(6)(7)(43)(44)(45)(46)(47) (31,48,49) and D20 (31,48) (4, 7). In making the assignments it was assumed that the order of 'IC resonances of the hormones was the same as in the acyclic nonapeptide for all residues except cysteine.…”

“…Conformational analysis of data from proton nuclear magnetic resonance ('H NMR) spectroscopy has contributed significantly to elucidation of the preferred solution conformations of oxytocin (1,2) and lysine vasopressin ([Lys8]VP) (3)(4)(5)(6)(7). Nevertheless, the conformational analysis involves several approximations: (1) the relationship between the dihedral angle 0 (NH-CaH) and the measured three-bond coupling constant 3JHNCH (8,9)-a spectral parameter most useful when considered in combination with potential energy maps (10,11); (2) temperature (12) and solvent (13,14) dependences of chemical shifts and proton exchange rates (15)(16)(17) of peptide hydrogens as related to their solvent-exposed or solvent-shielded state.…”