Ligand-binding properties of estrogen receptor proteins after interaction with surface-immobilized Zn(II) ions: evidence for localized surface interactions and minimal conformational changes.

摘要: The site- or domain-specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification purification receptor-associated regulatory components nucleic acids. We have demonstrated previously that restricted surface regions estrogen protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in at stationary phase metal ion interface were evaluated by monitoring alterations equilibrium dissociation constant (Kd) [3H]estradiol. Soluble (unliganded) present immature calf uterine cytosol via surface-exposed Zn(II)-binding to beads agarose derivatized iminodiacetate (IDA)-Zn(II) IDA-Zn(II) bound was incubated increasing concentrations [3H]estradiol (0.01–20 nM) presence absence unlabeled competitor (diethylstilbestrol) determine level specific hormone binding. Steroid-binding experiments performed parallel identical aliquots soluble receptor. Analyses data revealed a single class high-affinity (Kd = 2.44 ± 1.5 nM, n 10) steroid-binding which only marginally affected bindings 2.58 0.56 4). These are consistent location accessible site(s) on near DNA domain which, upon occupancy, do not influence domain. ions lack evident suggest phases loaded may be useful as tools analysis macromolecules.