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. Study holds ProTherm entries: 5705, 5706, 5707, 5708, 5709, 5710, 5711, 5712 Extra Details: intermolecular interactions; precursor; stability;,conformational change
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:30 p.m.
|Number of data points||16|
|Proteins||BETA-MERCAPTOPROPIONATE-OXYTOCIN ; Oxytocin-neurophysin 1|
|Assays/Quantities/Protocols||Experimental Assay: m pH:8.0 ; Experimental Assay: dG pH:8.0 ; Experimental Assay: m pH:10.0 ; Experimental Assay: dG pH:10.0 ; Experimental Assay: m pH:8.0 ; Experimental Assay: dG pH:8.0 ; Experimental Assay: m pH:6.0 ; Experimental Assay: dG pH:6.0|
|Libraries||Mutations for sequence YIQNCPLG|