5273
Side Chain Torsional Angles and Rotational Isomerism of Oxytocin in Aqueous Solution Herman R. Wyssbrod,*lbvcAlberto Ballardin,la*bI. L. Schwartz,lb Roderich Walter,IbGeorges Van Binst,'a William A. Gibbons,IC William C. Agosta,Ic F. H. Field,Ic and David Cowburn*IC Contributionfrom the Department of Organic Chemistry, Vrije Universiteit, Brussels, Belgium; the Department of Physiology and Biophysics, Mount Sinai Medical and Graduate Schools of the City University of New York, New York, New York 10029; and The Rockefeller University, New York. New York 10021. Received February I I 1977 ~
Abstract: Proton N M R spectra of oxytocin in aqueous solution over the temperature range 0-60 OC were measured, and the vicinal coupling constants and chemical shifts of a and fl protons and their temperature dependencies were derived. The possible fixed dihedral angles of the Cm-CS bonds in the cystyl region of the ring and the populations of several interconverting staggered rotamers were calculated. The small temperature dependencies suggest that there are no marked changes in conformation over the temperature range studied. These results add to our knowledge of the dynamic conformation of oxytocin in solution and are useful in considering possible biologically active conformers.
The conformation of the peptide hormone oxytocin is believed to be relatively flexible in aqueous solution.*The number of conformers contributing to the overall dynamic conformation is probably small, however. This latter assertion is based on 'H NMR studies of the conformation of oxytocin in diCY.1
- Tyrl
I
'k3
S I
S
Ay*6
An4
-
Oxytocin
b"5'
;ro7
- Gly9 - m2 methyl s~lfoxide,~ on calculation of conformations based either on energy minimization or on statistical analysis of the dependence of conformation on adjacent residues: on the relative rigidity of derivatives of o ~ y t o c i non , ~ the analysis of 2H and I3C relaxation times of oxytocin,6 and on the apparent Stokes radius of the m ~ l e c u l e If . ~there is indeed a small number of interconverting conformers, then it is plausible that in the 20-membered ring formed by the first six residues only a limited number of the torsional angles show rotational isomerism. I n addition, it is probable that rotational isomerism of side chains pendent to the ring will be restricted. Some information about values of torsional angles X I about the Ca-C@bonds and their rotational isomerism on the N M R time scale may be obtained by measurement of the vicinal coupling constants about these bonds for the side chains of individual residues in the peptide.* Two of these side chains, those of the half-cystyl residues, are part of the 20-membered ring. We have derived those Ha-H@coupling constants which may be obtained from 270-MHz proton spectra of the natural material without isotopic substitution over the temperature range 0-60 "C at pD 3.8. This paper summarizes the data obtained from these measurements and presents an interpretation of the vicinal coupling constants in terms of the conformation of the peptide.
Experimental Section Proton NMR spectra were collected on a Bruker HX-270 spectrometer. Sample concentration was 40 mg of peptide/mL of D20. An exponential decay equivalent to 0.4 Hz in the frequency domain was applied prior to the transformation of the free-induction decay (the average of about 64 transients) in order to increase the signalto-noise ratio at the expense of a slight loss in resolution. After Fourier transformation the 2703-Hzspectral width occupied 8 192 memory
locations. To compensate partially for the limited number of computer memory locations, peak positions were calculated from a three-point interpolation of a Lorentzian function. The pD was measured using an electrode equilibrated in DzO at 25 OC and calibrated using deuterated standards9 pD was adjusted to 3.8 using DCI. Sodium [2,2,3,3-2H4]-3-(trimethylsilyl)propionate (TSP) was added as an internal standard, from which all chemical shifts and resonance positions are reported in parts per million or hertz downfield. Analysis and simulation of spectra were performed by an implementation of LAOCN3,'' or analysis as an ABX spin system was performed in accordance with previously published In cases in which only the frequencies of the fl protons were observable, two possible solutions arise from the analysis. The solution used was selected on the basis that both vicinal couplings should be positive and
