Structure/Function of Recombinant Human Estrogen Receptor.
Annual rept. 1 Sep 94-31 Aug 95,
CALIFORNIA UNIV IRVINE
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The requisite initial step in estrogen receptor action, ligand binding, is mediated by the 250 amino acid Hormone Binding Domain HBD. We have expressed and purified in high yield approx 10 mgliter of culture two peptides amino acids 301-551 or 305-551 of the estrogen receptor in E. coil. Both peptides exhibit high affinity estradiol binding kd 0.3 nM and the ability to discriminate among various ligands comparable to the full-length protein. The isolated HBD 301-551 peptide displayed cooperative estradiol binding suggesting that it dimerizes and undergoes conformational changes required for cooperativity in a manner comparable to the full-length protein. In contrast, the HBD 305-551 peptide exhibited cooperativity only at protein concentrations at least 3-fold higher than those required for the longer peptide, suggesting that the N-terminus of the HBD contributes to dimer interactions. Both of the HBD peptides exhibited binding stoichiometries of 0.5 mol estradiolmol protein, suggesting that the HBD dimer binds a single molecule of estradiol. These results indicate that the use of fragments of the estrogen receptor expressed in E. coll lend insight into the structural features of the protein responsible for its functional properties.
- Medicine and Medical Research