Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons.
NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)
Live
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Rockefeller University, New York, New York 10021.
Cowburn
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Breslow
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D001127:Arginine Vasopressin; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009481:Neurophysins; D010121:Oxytocin; D011485:Protein Binding; D011487:Protein Conformation
D009481:Neurophysins; D001127:Arginine Vasopressin; D010121:Oxytocin