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

古斯塔夫斯·阿道夫学院的斯科特·布尔得到了化学系生命过程化学项目的一个奖项的支持，研究引起疟疾的恶性疟原虫。 该项目的重点是基因调控蛋白，含有一个分子识别位点称为溴结构域。 由于其独特的分子结构，布罗莫结构域能够识别对其他蛋白质（组蛋白）所做的特定分子变化，这些蛋白质参与细胞核内DNA的包装和储存。 组蛋白修饰可以强烈影响基因表达。 布罗莫结构域作为这种修饰的专门的“分子检测器”发挥作用。这项研究对通过开发寄生虫感染和癌症的潜在治疗方法来改善人类健康具有重要意义。 通过这个项目，基于发现的研究被纳入这个主要的本科院校（PUI）的课程。 参加课程活动的本科生获得实验设计，最先进的化学技术，并与其他科学家交流他们的研究经验。 这些技能可以转移到化学就业市场。关于特定的表观遗传调节蛋白在调节基因表达中所起的作用，特别是在非人类系统中所起的作用，人们知之甚少。 已知一种这样的调节蛋白，恶性疟原虫一般控制非阻遏蛋白5（PfGCN 5）的同源物，具有表观遗传调节作用。该项目旨在发现PfGCN 5布罗莫结构域优选的天然组蛋白乙酰化标记，并鉴定在其他布罗莫结构域存在下选择性破坏该结构域与其天然配体相互作用的合成配体。 NMR光谱技术用于观察PfGCN 5布罗莫结构域与模拟组蛋白尾部的肽的相互作用。这些组蛋白尾部含有特异性乙酰化的赖氨酸残基，使用固相肽合成产生。 在该项目的第二部分，PfGCN 5的高亲和力合成配体正在使用基于片段的配体发现策略进行鉴定。 使用连接或生长策略将片段翻译成具有更高亲和力的更大配体，然后通过计算机片段对接实验、PrOF NMR筛选和PfGCN 5溴结构域与结合片段的X射线晶体学分析来评估蛋白质-片段相互作用。 这项研究为理解溴结构域和乙酰化组蛋白之间的选择性相互作用的要求以及对选择性重要的分子识别事件提供了基础。 结构-活性研究很早就被纳入Gustavus Adolphus College的本科课程中，为本科生提供真实的研究经验和接触生物分子聚合物的生产，分离和分析技术。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.