Characterizing the Dynamic Response of the Estrogen Receptor to Agonists and Antagonists by Multifrequency Electron Spin Resonance Spin- Labeling

摘要

The overall objective of this project is to characterize the detailed structural and dynamic response of the estrogen receptor ligand binding domain (ER-LBD) to a variety of ligands ranging from strong estrogens to strong antiestrogens using electron spin labeling. The first two technical aims for this reporting period involved completing preparation of site-directed spin-labeled mutants of the ER-LBD and completing synthesis new spin-labeled ligands for the proposed studies. These tasks have essentially been completed and led to the development of a new fluorescence and EPR-based ER ligand binding assay. Towards the third technical aim of the reporting period, we have completed several initial EPR studies of ligand-dependent dynamics as a function of position in the ER as well as a series of spin-spin distance measurements. The results clearly support our initial hypothesis that the physical response of the ER protein to different ligand types can be resolved and characterized by EPR spin-labeling.