Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01178 (oxytocin)
 15,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects on bovine neurophysin-I of binding the perdeuterated peptides Phe-PheNH2 and Leu-PheNH2 were compared by proton NMR. A unique difference between the two peptides in their effects on Tyr-49 ring protons indicated proximity of the Tyr-49 ring to the side-chain of position 1 of bound peptide. Non-deuterated oligopeptides containing Phe in position 3 and no methyl groups induced different changes in neurophysin methyl resonances than dipeptides, suggesting shielding of one or more protein methyl groups by Phe-3. The results demonstrate that the identity of neurophysin residues at the hormone-binding site can be probed by analysis of changes induced in the protein spectrum by systematically related NMR-transparent peptides.
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PMID:Use of perdeuterated peptides in NMR studies of neurophysin-hormone interaction: demonstration of peptide-specific changes in neurophysin resonances. 400 58

The contribution of intramolecular hydrogen bonding to the solution structure of oxytocin was evaluated by study of amide hydrogen exchange rates in D2O by Fourier transform 1H NMR spectroscopy. Resolution enhancement filtering was employed in the determination of individual pseudo-first-order rate constants. Apparent barriers to exchange of 0.5 and 0.6 kcal mol-1 were measured for Asn5 and Cys6 peptide NH, respectively. The slowing is best explained by steric hindrance to solvent access in the case of Asn5, while for the Cys6 participation in a weak intramolecular hydrogen bond is possible. Fourfold acceleration of base-catalyzed exchange was observed for Tyr2 NH; it is proposed that this is the result of electronic effects induced by hydrogen bonding of Cys1 C=0, either to Cys6 NH or to the N-terminal amino group. Exchange proceeds near the random coil limit for each of the remaining residues. Comparison with exchange data for the model tripeptide N-acetyl-L-prolyl-L-leucylglycinamide demonstrates no evidence of noncovalent association of the tocin ring with the tripeptide tail of the hormone.
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PMID:Hydrogen--deuterium exchange kinetics of the amide protons of oxytocin studied by nuclear magnetic resonance. 626 Jan 37

The proton NMR spectra and role in peptide binding of carboxyl-terminal and NH2-terminal neurophysin residues were studied by preparation of bovine neurophysin-I derivatives from which residues 90-92 had been cleaved by carboxypeptidase or residues 1-8 excised by trypsin. The carboxypeptidase-treated protein showed normal peptide-binding behavior. NMR comparisons of this derivative and the native protein allowed identification of proton resonances associated with residues 89-92, confirmed a lack of functional role for this region of the protein, and permitted new observations on the behavior of neurophysin's aromatic residues. The trypsin-treated protein bound peptide with an affinity only 1/50 that of the native protein at pH 6 but evinced the same binding specificity and pH dependence of binding as the native protein. These results argued against direct interaction of residues in the 1-8 sequence with bound peptide and for a role for these residues, particularly Arg-8, in conformational stabilization of the active site; this role is held to be additional to the reported influence of 1-8 on dimerization. NMR comparisons of the trypsin product and native protein allowed preliminary assignment of a set of alkyl proton resonances to residues within the 1-8 sequence and were compatible with a restricted environment for Arg-8. Conformational differences between native and trypsin-treated proteins were manifest particularly by differences in the NMR spectra of Phe and Tyr-49 ring protons. The behavior of Phe ring protons was consistent with the reported decreased dimerization constant of the trypsin product and suggested participation of Phe-22 or -35 in dimerization. The behavior of Tyr-49 provided the first direct evidence of a change in secondary or tertiary structure associated with excision of residues 1-8. Suggested mechanisms by which this conformational change reduces binding include a direct effect on Tyr-49 and/or a conformational rearrangement of active site residues near Tyr-49.
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PMID:Proton magnetic resonance and binding studies of proteolytically modified neurophysins. 670 74

Carbon-13 NMR was used to study the interaction of the hormone oxytocin with neurophysin (NP). Oxytocins specifically enriched to 90% 13C in the alpha-carbons of Leu-3 (in [3-leucine]oxytocin), Gln-4, and Leu-8 and in the carbonyl carbon of Cys-6 were synthesized, so that the effect on these positions of binding to NP could be monitored. The alpha-carbons of residues 3 and 4 experienced shifts of -4.2 and -1.5 ppm (negative shifts are downfield), respectively, upon binding of the hormone to NP. The carbonyl carbon of residue 6 underwent a shift of +0.7 ppm, while the alpha-carbon of residue 8 displayed no shift. For each enriched residue, the hormone diastereoisomer in which this residue had the D configuration was also synthesized. NMR was then used to determine the binding affinity of the various diastereoisomers to NP, as well as to measure the NMR parameters of the bound peptides. When position 3 had the D configuration, the binding affinity for NP was 10-20% that of the native hormone. For positions 4, 6, and 8, the D diastereoisomers bound with the same affinity as oxytocin. The alpha-carbons of D residues of positions 3 and 4 shifted by -2.5 and +0.4 ppm, respectively, the carbonyl carbon of D-Cys-6 shifted by +1.4 ppm, and the alpha-carbon of D-Leu-8 was unshifted on binding to NP. The shift and diastereoisomer binding data, combined with previous results involving enriched carbons and/or diastereoisomers of residues 1, 2, and 9, support the conclusion that residues 1 and 2 are most crucial for binding of oxytocin to NP, residue 3 is less important, and residues 4-9 are of only slight significance.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Carbon-13 chemical shifts on oxytocin as a consequence of its interaction with neurophysins. 673 77

The synthesis of two spin-labels capable of binding to the hormone-binding site(s) of neurophysin is described. The two spin-labels are 4-(glycyl-L-phenylalanylamido)-2,2,6,6-tetramethylpiperidinyl-1-oxy and S-[[[3-(2,2,5,5-tetramethylpyrrolidine-1-oxy)amino]carbonyl]methyl]-L-cysteinyl -L-tyrosine amide; synthesis of the former is achieved by a novel route to circumvent problems associated with nitroxide instability under standard conditions of peptide deblocking. NMR studies of the effects of binding these spin-labels on relaxation rates of individual proton resonances of neurophysin were used to calculate correlation times and distances between the bound nitroxides and the observed protons. The results indicate that residue 3 of peptides bound to the strong site of neurophysin is greater than or equal to 14 A from Tyr-49 and argue against a distance of < 5 A between the ortho ring protons of Tyr-49 and those of residue 2 of peptides bound to the strong site. Alternatively, the data suggest that the previously observed nuclear Overhauser effect between these protons reflects spin diffusion at the strong site and a contribution of uncertain magnitude from a second but very weak binding site; this second site is close to Tyr-49 and is detected by the increased relaxation rate of Tyr-49 ring protons when 4-(glycyl-L-phenylalanylamido)-2,2,6,6-tetramethylpiperidinyl-1-oxy is displaced from the strong site by competing diamagnetic peptide. Additionally, the data indicate that residue 3 of bound peptides at the strong site is distant from His-80 but approximately 12 A from the amino terminus. The extended side chain of residue 1 of peptides at the strong site is calculated as less than or equal to 10 A from Tyr-49.
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PMID:Synthesis of peptide spin-labels that bind to neurophysin and their application to distance measurements within neurophysin complexes. 745 33

The primary structure of an elephant neurophysin, homologous to vasopressin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer-monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tyr-75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and/or hydrogen bonding. The Tyr-75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr-49, distant from the monomer-monomer interface, but adjacent to the peptide-binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75-78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization-mediated conformational changes that alter the binding site in the unliganded state.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amino acid sequence and properties of vasopressin-associated elephant neurophysin. 782 4

The NMR behavior of the aromatic protons of bovine neurophysin-I and its complexes was interpreted with reference to the 2.8 A crystal structure of the dipeptide complex of bovine neurophysin-II and to mechanisms underlying the thermodynamic linkage between neurophysin dimerization and peptide binding. Large binding-induced shifts in the ring proton signals of Tyr-2 of ligand peptides (approximately 0.5 ppm upfield and approximately 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other investigators, evidence is presented indicating the absence of dipolar contact between Tyr-2 ring protons and protein Phe ring protons. The large binding-induced shifts are attributed to a strong influence of proximal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the behavior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interface appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptide binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface. Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participant, were demonstrated to be markedly altered both by dimerization in the unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively support a model in which conformational differences between unliganded monomer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domain segments of the interface, which are homologous, are affected differently by peptide binding.
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PMID:NMR behavior of the aromatic protons of bovine neurophysin-I and its peptide complexes: implications for solution structure and for function. 785 24

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.
J Biomol NMR 1993 Nov
PMID:Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide. 811 Dec 31

The first crystal structure of the pituitary hormone oxytocin complexed with its carrier protein neurophysin has been determined and refined to 3.0 A resolution. The hormone-binding site is located at the end of a 3(10)-helix and involves residues from both domains of each monomer. Hormone residues Tyr 2, which is buried deep in the binding pocket, and Cys 1 have been confirmed as the key residues involved in neurophysin-hormone recognition. We have compared the bound oxytocin observed in the neurophysin-oxytocin complex, the X-ray structures of unbound oxytocin analogues and the NMR-derived structure for bound oxytocin. We find that while our structure is in agreement with the previous crystallographic findings, it differs from the NMR result with regard to how Tyr 2 of the hormone is recognized by neurophysin.
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PMID:Crystal structure of the neurophysin-oxytocin complex. 856 43

The active site of liganded neurophysin contains a salt bridge that involves the side chains of Arg-8 and Glu-47 of the protein and the alpha-amino group of bound hormone or related peptide. The extent to which the Arg-8-Glu-47 salt bridge persists in the absence of peptide, or to which the environment of Arg-8 in the unliganded state differs in monomers and dimers, is relevant to an understanding of allosteric mechanism in this system. In the present study, the behavior of the salt bridge was investigated by 15N NMR and chemical replacement of Arg-8. Bovine neurophysin-I was converted to its des 1-8 derivative, and Arg-8 was replaced by 15N-substituted Arg or by other residues using chemical semisynthesis. The relative abilities of different amino acids to restore peptide affinity to the des 1-8 protein were in good accord with the view of the salt bridge in the liganded state obtained from crystals of bovine neurophysin-II complexes. In the unliganded state, comparison of the 15N and proton NMR signals from Arg-8 with those in smaller arginine systems suggested the absence of significant interactions between the guanidinium of Arg-8 and Glu-47 or between the amino terminal region of Arg-8 and other elements of the protein. No evidence of a difference in Arg-8 environment between unliganded monomers and dimers was found. Marked spectral changes accompanying the binding of oxytocin indicated changes in the environment of both the side chain and amino terminal region of Arg-8. The NMR results were in good agreement with a recently emerging comparison of bovine neurophysin-II derivatives in the liganded and unliganded states, with the notable exception of the extent of salt bridge formation in the unliganded state. The results are shown to be consistent with, and to help explain, significant differences between the two bovine neurophysins in the susceptibility to tryptic cleavage at Arg-8 in the unliganded state and in the pH dependence of peptide binding and additionally constrain potential allosteric mechanisms underlying neurophysin ligand-facilitated dimerization.
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PMID:The behavior of the active site salt bridge of bovine neurophysins as monitored by 15N NMR spectroscopy and chemical substitution. Relationship to biochemical properties. 879 57


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