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Fluorine NMR studies of the human estrogen receptor

The estrogen receptor is a member of the steroid receptor superfamily that includes proteins whose functions are to bind small hydrophobic ligands and modulate transcriptional activity within the nucleus. The hormone binding domain (HBD) located on the C-terminus of the protein is responsible for the high affinity binding of ligands which mediate the biological responses of the hormone. There has been a wide interest in the action of estrogen and estrogen-like materials with the receptor since a host of disease states including Breast Cancer, osteoporosis and endometriosis have been associated with altered production of steroids. We need to understand the molecular basis for the function of the estrogen receptor so we can monitor its action. Since HBD is responsible for high-affinity binding of steroid and undergoes conformational changes that may be the key point in nuclear transcription events, our laboratory has undertaken studies to probe ligand binding and subsequent conformational changes in HBD by F-19 NMR. F-19 NMR has been proven to be a useful tool in the study of structure and dynamics in protein systems too large for conventional NMR methods since the nucleus is easily incorporated at specific sites within the protein or ligand where it provides a nonperturbing probe without background signals. We describe herein three systems that were developed to yield high level expression of soluble HBD with fluorine labels in E. coli. To further characterize the HBD we have evaluated the fluorinated derivatives of diethylstilbestrol (DES). Our preliminary work has shown the applicability of the fluorine nucleus to probe ligand binding and conformational change in the steroid receptor superfamily.

estrogen; steroid; receptor; diethylstilbestrol; F-19 NMR spectroscopy; fluorine-labeled protein; protein structure; protein conformation

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