“…The data therefore indicate that the intrinsic affinity of a dimer site is minimally 10 times greater than that of the monomer under these conditions, in turn indicating a minimum 100-fold increase in the dimerization constant for the fully liganded state relative to the unliganded state. This value can be compared with the peptide-induced 130-fold increased association of bovine neurophysin I with neurophysin II and the ~35-fold increased self-association of bovine neurophysin II measured by affinity chromatography (Abercrombie et al, 1984;Kanmera & Chaiken, 1985;Fassina & Chaiken, 1988). Sedimentation equilibrium patterns of native neurophysin II (Nicolas et al, 1975) and nitrated neurophysin I (Nicolas et al, 1978) also suggest at least a 100-fold increase in the dimerization constant in the presence of bound peptide hormone.…”
The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.
“…The data therefore indicate that the intrinsic affinity of a dimer site is minimally 10 times greater than that of the monomer under these conditions, in turn indicating a minimum 100-fold increase in the dimerization constant for the fully liganded state relative to the unliganded state. This value can be compared with the peptide-induced 130-fold increased association of bovine neurophysin I with neurophysin II and the ~35-fold increased self-association of bovine neurophysin II measured by affinity chromatography (Abercrombie et al, 1984;Kanmera & Chaiken, 1985;Fassina & Chaiken, 1988). Sedimentation equilibrium patterns of native neurophysin II (Nicolas et al, 1975) and nitrated neurophysin I (Nicolas et al, 1978) also suggest at least a 100-fold increase in the dimerization constant in the presence of bound peptide hormone.…”
The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.
“…The presence of dimer signals and the absence of monomer signals are consistent with the higher dimerization constant reported for both the semisynthetic precursor and the noncovalent mature hormone−NP complex ( 9 , 10 , 32 ) relative to that of the unliganded protein. If a 100-fold higher dimerization constant of the precursor relative to unliganded protein is assumed ( , ), the weight percent of dimer at this protein concentration (0.2 mM) should exceed 95%. 8 Comparison of the 400 MHz NMR spectrum of the precursor with that of the 1/1 complex of bovine NP-I and oxytocin at pH 6 in D 2 O.…”
Section: Resultsmentioning
confidence: 99%
“…A central conclusion of their work was that interactions between hormone and NP segments within the precursor were analogous to those within processed hormone-NP complexes, and conferred on the precursor self-association properties analogous to those of the complexes. The precursor was also demonstrated to be more stable than unliganded NP (9), but the additional stability was not quantitated.…”
mentioning
confidence: 99%
“…Such properties are of particular interest in view of the importance of the precursor to the proper targeting of hormone to regulated neurosecretory granules and the failure of such targeting in diabetes insipidus, a disorder characterized by insufficient vasopressin production ( , ). Chaiken and co-workers ( , ) prepared the bovine oxytocin precursor by semisynthesis, chemically ligating the hormone-containing segment to biologically derived bovine NP-I. A central conclusion of their work was that interactions between hormone and NP segments within the precursor were analogous to those within processed hormone−NP complexes, and conferred on the precursor self-association properties analogous to those of the complexes.…”
Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.
“…The neurophysins exhibit hormone-enhanced self-association as well as hormone binding that is enhanced by self-association (Breslow et al, 1971;Chaiken et al, 1975;Nicolas et al, 1976Nicolas et al, , 1980 Pearlmutter & Dalton, 1680; Tellam & Winzor, 1980;Angal & Chaiken, 1982). The interactions of the neurophysins and hormones have been evaluated previously by analytical affinity chromatography on nonrigid, agarose-based affinity matrices (Angal & Chaiken, 1982; Abercrombie et al, 1984; Kanmera & Chaiken, 1985). Consequently, this neuroendocrine peptideprotein system provides an excellent model for evaluating the usefulness of analytical high-performance affinity chromatography as a methodology for characterizing macromolecular interactions.…”
Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)
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