Using the human estrogen receptor (ER) as a model system, the proposed CAREER project focuses on three specific aims: (1) To develop a novel directed evolution strategy - in vitro co-evolution to create novel ER ligand specificity. (2) To engineer an orthogonal ligand-receptor pair based on the ER ligand binding domain and a synthetic chemical using a combined directed evolution and rational design approach and subsequently construct a genetic switch. (3) To use various biochemical and biophysical methods including DNA sequencing, transactivation assay, ligand binding affinity assay, molecular modeling and molecular docking to characterize all the evolved ER mutants resulting from the above two specific aims in an effort to uncover the molecular bases for ER ligand specificity and the molecular evolution mechanisms of nuclear receptors. Such studies will provide insights into the pharmacology of the ER and assist in the development of estrogen pharmaceuticals for applications such as menopausal hormone replacement therapy and breast cancer treatment. The resulting findings will also be invaluable for the studies of other types of nuclear receptors, most of which are linked to various diseases and represent major drug targets. The proposed educational development plan comprises two components: an on-campus research and education program in biomolecular engineering, and a biotechnology outreach program.
