In this project the PI will characterize the first steps of gene activation by nuclear receptors. Nuclear receptors are proteins with the ability to regulate the expression of adjacent genes and these receptors are classified as transcription factors. The PI will investigate the synergistic ligand effect observed for a hetero-dimeric nuclear receptor by examining its interaction with two cofactor proteins. The PI will use three-color brightness analysis to study ternary protein complexes directly inside living cells. The proposed studies will inspire further applications of three-color brightness analysis to other complex biological problems. The PI will provide research opportunities for REU students during the summer, which will expose these students to biological physics and encourage their interest in this subject. In addition, the PI will organize an annual three-day hands-on work shop for students from the biological sciences. The workshop will provide needed training for students who apply fluorescence techniques in their daily research, but without necessarily having a detailed understanding of the underlying principles.
Protein molecules are critical for carrying our all essential processes inside the living cell, though their action may not be easily observed. Proteins are not only colorless, but also too small to be seen in an optical microscope. To overcome this challenge special fluorescent proteins are available that can be genetically linked to a colorless protein, thereby labeling it and making it visible. Every time a labeled protein passes through a laser beam it responds by giving off a minute amount of light with a specific color that depends on the fluorescent protein it is linked to. Proteins do not act alone, but associate with other proteins to form protein complexes that carry out function. Observing these protein complexes in cells is important, because it provides important clues how cells function. By labeling two proteins with different colors it is possible to optically observe protein association within the living cell. A protein complex carrying both a green and red label generates light containing both colors when passing through a small laser focused inside the cell, but only a single color is generated when a single protein enters. This concept can be turned into a quantitative tool by statistical analysis of the detected light, which we refer to as brightness analysis. The research supported by this award was designed to extend brightness analysis to the interactions of three different proteins. Interactions between three proteins are especially interesting, because biology can use the association of two proteins to change its shape so that the binding of the third protein becomes easier or harder. This mechanism provides a clever way to regulate cells. After building the instrument we identified three fluorescent proteins that are suitable labels for three-color brightness experiments. We also developed the necessary analysis tools and tested their performance by measuring well-characterized model protein systems. Once these steps were completed we applied the technique to characterize the interaction of three cellular proteins and observed the formation of a ternary protein complex. We further were able to detect the remodeling of the ternary protein complex in response to a small hormone added to the cell culture. We demonstrated that quantitative three-color brightness studies in cells are feasible and hope that the technique will mature into a powerful tool for the characterizations of ternary protein complexes in living cells.
