Binding and spectroscopic properties of ostrich neurophysins. Probing the role of residue 35 at the monomer-monomer interface.
Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr-35, a residue that provides a potential probe of the monomer-monomer interface and of allosteric interrelationships between this region and the binding site. Mesotocin-associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel-filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr-35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non-denaturing conditions. Reaction of Tyr-35 of the mesotocin-associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr-35 to an unusual 237 nm band in the mesotocin-associated protein. Small shifts in the 350 nm CD band of nitrated Tyr-35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr-35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin-associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.
Breslow
E
E
Department of Biochemistry, Cornell University Medical College, New York, NY.
LaBorde
T
T
Saayman
H S
HS
Oelofsen
W
W
Naudé
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001717:Birds; D002417:Cattle; D002850:Chromatography, Gel; D008969:Molecular Sequence Data; D009481:Neurophysins / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods; D014443:Tyrosine / Q000737:chemistry
D009481:Neurophysins; D014443:Tyrosine