| dc.description.abstract | Estrogen receptor alpha (ER) and BRCA1 play an important role in the development of breast cancer, and multiple pathways link these two proteins together. Previous studies have identified the ligand binding domain (LBD) of ER and residues 1 through 258 of BRCA1 as important in the direct physical interaction between these two proteins. This study aimed to characterize the binding kinetics of this interaction in the presence and absence of 17-estradiol (estrogen) with a shortened BRCA1 construct (residues 177-258); however, binding between ER LBD and this BRCA1 construct could not be detected through fluorescence emission spectroscopy or isothermal titration calorimetry (ITC). Synthesizing ER LBD presented challenges with low yield, so the purification protocol was refined to cool bacterial cultures at an OD600 of 0.2 during growth and add dithiothreitol during lysis for improved yield. A 24% decrease in fluorescence intensity upon addition of estrogen to ER LBD confirmed the ligand-binding functionality of the protein. Additionally, Stern-Volmer studies verified that the estrogen binding site on ER LBD is located in close vicinity to the tryptophan residues in the protein since fluorescence quenching was more efficient in the absence of estrogen. Finally, factors contributing to the absence of ER-BRCA1 binding are discussed, including the length of the BRCA1 construct used or the potential necessity of an additional protein, BARD1. | |
