INTERACTION OF OXYTOCIN WITH CA2-RESONANCE AND MOLECULAR MODELING STUDIES OF CONFORMATIONS OF THE HORMONE AND ITS CA2+ COMPLEX( .2. PROTON MAGNETIC)

Drastic changes in the CD and fluorescence spectra of oxytocin [cyclo( Cys(1)-Tyr(2)-lle(3)-Gln(4)-Asn (5)-Cys (6)) -pro(7)-Leu(8)-Gly(9)-NH2 ] occur on binding Ca2+ in trifluoroethanol (Ananthanarayanan and Brim ble, preceding paper). To further characterize the conformation of the Ca2+-bound hormone, we carried our H-1-nmr measurements in deuterated trifluorethanol of oxytocin and its 1 : 1 Cn(2+) complex. The one-dim ensional mnr, data identified residues involved in Ca2+ binding and th e extent of their perturbation on Ca2+ addition. The (3)J(NH-CH) coupl ing constants and two-dimensional nuclear Overhauser effect (NOE) spec tral cross peaks confirmed the helical nature of the Ca2+ complex dedu ced from CD data. lnterproton distances in the flee hormone and its Ca 2+ complex were estimated from the respective NOE data. Apparent globa l minimum-energy conformations of free and Ca2+ bound oxytocin were co mputed using the Monte Carlo with energy minimization protocol, with a nd without incorporating the NOE-derived distance constraints. Taken t ogether, our results show Ca2+ binding to oxytocin to be a two-step pr ocess. The binding of the first Ca2+ brings the otherwise extended tai l segment of oxytocin closer to the ring moiety so that it wraps aroun d the cation. This cause the maximal extent of change in all the spect ral paramerers. The subsequent formation of the I Ca-oxytocin complex results in the tail detaching itself away from the ring so as to bind the second Ca2+ ion. This lends to further spectral changes in the hor mone molecule. The tail segment plays a major role in both steps. Thes e observations may be useful in understanding the structural basis of oxytocin action. (C) 1997 John Wiley & Sons, Inc.