Conformational flexibility of neurophysin as investigated by local motions of fluorophores. Relationships with neurohypophyseal hormone binding

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Abstract
Flexibility of various structural domains of neurophysin and neurophysin-neurohypophyseal hormone complexes was investigated through the fast rotational motion of fluorophores in highly viscous medium. Despite 7 intrachain disulfide links, some domains of neurophysin remain highly flexible. Dimerization of neurophysin does not affect the structural integrity of the individual subunits, each subdomain being conformationally equivalent within each protomer of the unliganded dimer. The absence of heterogeneous fluorescence anisotropy precludes the existence of a dimer tautomerization equilibrium. Binding of the hormonal ligands to neurophysin dimer promotes a large conformational change over the whole protein structure as assessed by differential alterations of the flexibility-rigidity and intrasegmental interaction properties of domains that do not participate directly to the dimerization/binding areas. The order of free-energy coupling between ligand binding and protein subunit association was evaluated. The 1st mole of bound ligand stabilizes the dimer by increasing the intersubunit contacts while the 2nd mole of ligand induces most of the described conformational change. The positive cooperativity between the 2 dimeric binding sites is linked mainly to the binding of the 2nd ligand. The induced structural change is perceived differently by each subunit as assessed by opposite local motions of Tyr49 in each liganded protomer and leads to the formation of a dimeric complex with a global pseudospherical symmetry although containing domains of local asymmetry.