Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide

Abstract

With the combined use of various two-dimensional (2D) NMR techniques, a complete assignment of the 1H and 13C resonances of oxytocin, Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2, for two molecular states, protonated and unprotonated at the N-terminal group, was performed in dimethyl sulfoxide. A small but distinct change in the backbone conformation of the six-residue cyclic moiety, associated with the protonation, was first suggested from those NMR parameters relevant to conformation, such as change with temperature in the chemical shifts of the peptide amide protons and changes in chemical shifts and homonuclear as well as heteronuclear three-bond coupling constants. The solution structures of oxytocin for the protonated and unprotonated forms were then calculated using distance analysis in dihedral-angle space, based on a relaxation matrix evaluated from quantitative NOE intensities at different mixing times. Total amounts of 93 and 105 distances were determined for the protonated and the unprotonated forms, respectively. There were 25 interresidue distances relevant to the structure of the cyclic moiety for the protonated form of oxytocin and 43 for the unprotonated form. Overall structures with the lowest target penalty function were similar between the two forms, having a beta-turn structure at the endocyclic residues of the Tyr-Ile-Gln-Asn moiety. The local backbone conformations near the N-terminus, however, were significantly different between the two forms. This was found to be due to a change in the dihedral angle of the disulfide bridge (chi ss around C-S-S-C), which closes the ring in the cyclic peptide. The dihedral angle was about +90 degrees for the unprotonated form and an intermediate value of about +45 degrees for the protonated form.

Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR

“…The polarization-transfer matrix, R, 30 ϫ 30 in size, was determined with a FORTRAN program for the nonlinear leastsquares method by using the Levenberg-Marquardt algorithm. The initial R was estimated linearly from experimental signal intensities with a short mixing time (25 where F j () is the single-quantum (SQ) dipolar lineshape function of spin j under CH couplings. The linewidths for F j () were determined primarily from the experimental SQ lineshapes obtained with a 2D chemical-shift resolved CH dipolar spectrum (26).…”

“…Experimental peak intensities are caused by both direct and relayed polarization transfer mechanisms, but R gives the rates caused by the direct dipolar couplings (25). Thus the determination of R provides the distance information only from direct dipolar couplings.…”

Complete NMR analysis of oxytocin in phosphate buffer

“…Previous conformational studies of OXT by nuclear magnetic resonance (NMR) have been readily performed in solvents such as dimethyl sulfoxide (DMSO) [2 -5] and have shown that the assignment of almost all of the 1 H and 13 C resonances of OXT can be obtained in DMSO solvent. [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.…”

Determination of a Binding Site of Cu and Ni Metal Ions with Oxytocin Peptide by Electrospray Tandem Mass Spectrometry and Multiple Mass Spectrometry

“…2,4 two b-turn structures were also reported on the basis of the results of NMr spectroscopy experiments involving oxytocin. [5][6][7] A first b-turn structure was proposed in a cyclic cys1-tyr2-Ile3-Gln4-Asn5-cys6 pentapeptide involving the sequence tyr2-Ile3-Gln4-Asn5, and a second b-turn structure was proposed in the c-terminal sequence cys6-Pro7-Leu8-Gly9.…”

“…[5][6][7] A first b-turn structure was proposed in a cyclic cys1-tyr2-Ile3-Gln4-Asn5-cys6 pentapeptide involving the sequence tyr2-Ile3-Gln4-Asn5, and a second b-turn structure was proposed in the c-terminal sequence cys6-Pro7-Leu8-Gly9.the structure of oxytocin complexed with a receptor 8-9 and the biological activities of [metal ion---oxytocin] complex [10][11][12][13][14][15][16][17] have been studied by many research groups. …”