RUI: Applications of 19F NMR spectroscopy to evaluate non-human bromodomain molecular recognition

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Scott Bur of Gustavus Adolphus College is supported by an award from the Chemistry of Life Processes Program in the Division of Chemistry to study the malaria-causing parasite Plasmodium falciparum. The focus of the project is on gene-regulating proteins that contain a molecular recognition site known as the bromodomain. Because of its unique molecular structure, the bromodomain is able to recognize specific molecular changes made to other proteins---histones---that are involved in packaging and storing DNA within the cell nucleus. Histone modifications can strongly affect gene expression. The bromodomain functions as a specialized "molecular detector" for such modifications. This research has implications for improving human health by developing potential treatments for parasitic infection and cancer. Through this project, discovery-based research is incorporated within the curriculum of this primarily undergraduate institution (PUI). Undergraduate students participating in the course activities gain experience in experimental design, state-of-the-art chemical techniques, and communicating their research to other scientists. The skills are transferrable to the chemical job market. Little is known about the roles that specific epigenetic-regulator proteins play in regulating gene expression, especially in non-human systems. One such regulatory protein, the Plasmodium falciparum homolog of General Control Non-repressed protein 5 (PfGCN5), is known to have an epigenetic regulatory role. This project is aimed at discovering the natural histone acetylation mark preferred by the PfGCN5 bromodomain, and identifying synthetic ligands that selectively disrupt the interaction of this domain with its natural ligand in the presence of other bromodomains. NMR spectroscopy techniques are used to observe interactions of the PfGCN5 bromodomain with peptides that simulate histone tails. These histone tails, which contain specifically acetylated lysine residues, are produced using solid-phase peptide synthesis. In the second part of the project, high-affinity synthetic ligands for PfGCN5 are being identified using a fragment-based ligand discovery strategy. Fragments are translated into larger ligands with higher affinity using linking or growing strategies, and protein-fragment interactions are then assessed via in silico fragment docking experiments, PrOF NMR screening, and X-ray crystallographic analysis of the PfGCN5 bromodomain with a bound fragment. This research provides a foundation for understanding the requirements of selective interactions between the bromodomain and acetylated histone, and molecular recognition events that are important for selectivity. Structure-activity studies are embedded early in the undergraduate curriculum at Gustavus Adolphus College, providing undergraduate students with authentic research experiences and exposure to techniques for the production, isolation, and analysis of biomolecular polymers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.