Thesis Title: The Use of Differential Scanning Fluorimetry to Detect Protein-Ligand Interactions in Human ERα
Thesis Advisor: Jeff Reinking
Thesis Abstract: Estrogen receptors (ERs) are Nuclear Receptors (NRs) which control gene regulation, namely the transcription of genes that control growth, differentiation, and those that play a part in the female reproductive system. ERs can be activated by endogenously produced estrogens, namely estrone (E1), estradiol (E2) and estriol (E3). ERs can be deactivated by antagonists such as the anti-cancer drug tamoxifen. recent studies have shown that increased levels of ER can cause developmental, reproductive, and behavioral deformities in people. because of this, and past research, there has been a growing concern of the possible health risks caused by estrogen like compounds in the environment as well as in manufactures goods that could bind to ER, resulting in illnesses. this has urged people to better understand ER and its interactions with different ligands. The original goal of this research was to determine if a newly developed technique known as Differential Scanning Fluorimetry (DSF) can be used to detect and characterize protein-ligand interactions in the human ERα, and by extension, NRs in general.
This was tested by screening samples from a nuclear receptor ligand chemical library with a human estrogen receptor ligand binding domain (ERα LBD). These were combined with SYPRO orange dye to form 10µL reactions in a 96-well plate. Interactors of ERα stabilize the protein, hence a larger amount of energy would be needed to melt the protein, resulting in an increase in its melting. The three known interactors of ERα resulted in a higher melting temperature, suggesting that they were bound to ERα. Therefore, DSF can be used as a new method for identifying interactions among nuclear receptors and their ligands. During these experiments, a fourth compound was occasionally shown to interact with ERα; this compound is a fungal metabolite known as paxilline. In order to identify the conditions at which paxilline binds to ERα, ER was grown and screened in both reducing and non-reducing conditions. Under non-reducing conditions, paxilline appeared to have a high melting temperature, similar to those of the known interactors, suggesting that paxilline is interacting with ERα. No interaction is seen however under reducing conditions. Further experiments are being conducted to better understand the interaction between ERα and paxilline.