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

Gustavus Adolphus学院的Scott Bur得到了化学部生命过程化学项目的支持，以研究导致疟疾的寄生虫恶性疟原虫。该项目的重点是基因调控蛋白，它包含一个被称为溴域的分子识别位点。由于其独特的分子结构，溴结构域能够识别其他蛋白质-组蛋白--的特定分子变化，这些蛋白质参与了DNA在细胞核内的包装和存储。组蛋白修饰可以强烈影响基因的表达。溴域作为一种特殊的“分子探测器”进行这种修饰。这项研究对通过开发潜在的寄生虫感染和癌症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到这个主要是本科院校(PUI)的课程中。参加课程活动的本科生将获得实验设计、最新化学技术的经验，并与其他科学家交流他们的研究成果。这些技能可以转移到化学就业市场。人们对特定的表观遗传调节蛋白在调控基因表达中扮演的角色知之甚少，尤其是在非人类系统中。其中一种调节蛋白，恶性疟原虫总控制非抑制蛋白5的同源物(PfGCN5)，已知具有表观遗传调节作用。该项目旨在发现PfGCN5溴结构域偏爱的自然组蛋白乙酰化标记，并鉴定在其他溴结构域存在的情况下选择性地破坏该结构域与其天然配体相互作用的合成配体。核磁共振波谱技术被用来观察PfGCN5溴域与模拟组蛋白尾部的多肽的相互作用。这些含有特定乙酰化赖氨酸残基的组蛋白尾巴是通过固相多肽合成产生的。在该项目的第二部分，正在使用基于片段的配体发现策略来确定PfGCN5的高亲和力合成配体。通过连接或生长策略，片段被翻译成更大的配体，具有更高的亲和力，然后通过硅胶片段对接实验、核磁共振教授筛选和PfGCN5溴域与结合片段的X射线晶体分析来评估蛋白质-片段的相互作用。这项研究为理解溴结构域和乙酰化组蛋白之间选择性相互作用的要求以及对选择性至关重要的分子识别事件提供了基础。结构-活性研究很早就嵌入了古斯塔夫斯·阿道夫学院的本科课程中，为本科生提供真实的研究经验，并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，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.