RESUMEN
Drastic changes in the CD and fluorescence spectra of oxytocin [cyclo(Cys1-Tyr2-Ile3-Gln4-Asn5-Cys6)-Pro7-Leu8-Gly 9-NH2] occur on binding Ca2+ in trifluoroethanol (Ananthanarayanan and Brimble, preceding paper). To further characterize the conformation of the Ca(2+)-bound hormone, we carried out 1H-nmr measurements in deuterated trifluorethanol of oxytocin and its 1:1 Ca2+ complex. The one-dimensional nmr data identified residues involved in Ca2+ binding and the extent of their perturbation on Ca2+ addition. The 3JNH-CH coupling constants and two-dimensional nuclear Overhauser effect (NOE) spectral cross peaks confirmed the helical nature of the Ca2+ complex deduced from CD data. Interproton distances in the free hormone and its Ca2+ complex were estimated from the respective NOE data. Apparent global minimum-energy conformations of free and Ca2+ bound oxytocin were computed using the Monte Carlo with energy minimization protocol, with and without incorporating the NOE-derived distance constraints. Taken together, our results show Ca2+ binding to oxytocin to be a two-step process. The binding of the first Ca2+ brings the otherwise extended tail segment of oxytocin closer to the ring moiety so that it wraps around the cation. This causes the maximal extent of change in all the spectral parameters. The subsequent formation of the 2:1 Ca-oxytocin complex results in the tail detaching itself away from the ring so as to bind the second Ca2+ ion. This leads to further spectral changes in the hormone molecule. The tail segment plays a major role in both steps. These observations may be useful in understanding the structural basis of oxytocin action.
Asunto(s)
Calcio/metabolismo , Oxitocina/química , Oxitocina/metabolismo , Conformación Proteica , Calcio/química , Calcio/farmacología , Dicroismo Circular , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Método de Montecarlo , Unión Proteica , Protones , Trifluoroetanol/farmacologíaRESUMEN
Conformational features of nicardipine in acetonitrile, in the absence and presence of Ca2+, were investigated by one-dimensional NMR and difference absorption spectroscopy techniques. The data show that in acetonitrile solution the antiperiplanar form of nicardipine is dominant. The addition of Ca2+ to the drug solution caused marked changes in the difference absorbance spectra in the 200-400 nm region and in many of its 1H and 13C NMR resonances. The changes were most significant up to a ratio of 0.5 Ca2+:drug. Analysis of the binding data showed the predominant species to be a 2:1 drug:Ca2+ "sandwich" complex with an estimated dissociation constant of 100 microM at 25 degrees C. One-dimensional nuclear Overhauser effect (NOE) experiments revealed through-space connectivities in the drug before and after Ca2+ binding. These changes in conjunction with the changes in 1H and 13C chemical shifts suggest a structure in which the 4-aryl ring substitute of the pyridine moiety moves closer to the C3-side chain in the presence of Ca2+. This attraction is achieved via the chelation of the Ca2+ ion by the oxygen atoms in the m-NO2 of the aryl group and the COOCH2 group in the side chain of the dihydropyridine ring, and gives rise to a stable synperiplanar conformation. A preference for this conformation was also observed in the Ca2+ complex of nifedipine in acetonitrile as inferred from the rather limited NOE data obtained. Our study provides a detailed solution structure for nicardipine and also leads to a suggestion of a role for Ca2+ in the action of this and possibly other dihydropyridines.