The behavior of the active site salt bridge of bovine neurophysins as monitored by 15N NMR spectroscopy and chemical substitution. Relationship to biochemical properties

Changsheng Zheng, Sean M. Cahill, Esther Breslow

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 α-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 neurophysinII 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-11 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.

Original languageEnglish (US)
Pages (from-to)11763-11772
Number of pages10
JournalBiochemistry
Volume35
Issue number36
DOIs
StatePublished - 1996
Externally publishedYes

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Neurophysins
Nuclear magnetic resonance spectroscopy
Catalytic Domain
Substitution reactions
Magnetic Resonance Spectroscopy
Salts
Peptides
Nuclear magnetic resonance
Dimers
Monomers
Derivatives
Proteins
Dimerization
Guanidine
Oxytocin
Arginine
Protons
Hormones
Ligands
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

The behavior of the active site salt bridge of bovine neurophysins as monitored by 15N NMR spectroscopy and chemical substitution. Relationship to biochemical properties. / Zheng, Changsheng; Cahill, Sean M.; Breslow, Esther.

In: Biochemistry, Vol. 35, No. 36, 1996, p. 11763-11772.

Research output: Contribution to journalArticle

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abstract = "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 α-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 neurophysinII 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-11 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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AB - 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 α-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 neurophysinII 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-11 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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