Dissecting the Molecular Mechanisms of the Histone Acetyltransferase/Cyclic Adenosine Monophosphate Binding Protein Interactome Using Protein-Observed Fluorine NMR

生物系统中的蛋白质以一种“化学盲文”进行交流，其中一个蛋白质分子的分子尺度凸起和凹槽通过另一个蛋白质的物理相互作用来读取。详细了解这些蛋白质-蛋白质相互作用的分子水平通信对于确定控制正常和异常细胞功能的基本规则是必要的。在分子水平上表征和操纵这些相互作用的能力已被证明具有挑战性。明尼苏达大学的Pomerantz实验室致力于了解蛋白质-蛋白质相互作用的化学细节，其长期目标是开发出阻止导致有害细胞功能的蛋白质-蛋白质交流的方法。为了实现这一目标，新型基于氟的“探针”蛋白质分子报告了难以检测的蛋白质-蛋白质相互作用，因为它们在不同的化学环境中具有内在的响应性。通过以课程为基础的本科研究经验（CUREs），研究工作与教育活动相结合，旨在提高学生的成功，并增加在科学-技术-工程-数学职业道路上的保留率。通过与Gustavus Adolphus学院的合作，以及一个教育训练营，扩大了本科生接触化学生物学研究的机会，并由Pomerantz团队提供指导，扩大了研究项目的影响。美国国家科学基金会化学系生命过程化学项目资助明尼苏达大学双城分校的William Pomerantz博士开发氟核磁共振波谱（NMR）方法，用于检测、量化和定义转录因子蛋白-蛋白界面的新型相互作用模式，包括那些涉及表观遗传调节蛋白的模式。Pomerantz团队的长期研究目标是开发化学工具，以探索转录因子蛋白相互作用在表观遗传基因调控中的生物学作用。本研究将Pomerantz实验室开发的氟-19核磁共振方法与其他生物物理方法相结合，以表征天然和合成配体结合相互作用。本研究详细了解了参与蛋白质变构网络的芳香族氨基酸在调节CREB结合蛋白（CBP）的KIX蛋白-蛋白相互作用域中的作用。Pomerantz团队最近的一项发现是在合成小分子靶向的KIX结构域上发现了一个新的蛋白质相互作用位点。需要解决的一个关键的基本问题是其他蛋白质与新发现的小分子结合位点相互作用的性质。Pomerantz团队通过核磁共振对该位点进行了结构表征，并使用光交联和蛋白质组学方法来评估是否有其他蛋白质占据该结合位点，从而对KIX功能进行变变调节。他们评估潜在的域-域之间的相互作用，首先作为单独的域进行研究，最终作为全长结构进行研究。研究目标与创造基于课程的本科研究经验的教育目标相结合，这些研究经验来自于真实的本科化学学生研究经验，通过氟-19核磁共振方法研究蛋白质-配体相互作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Proteins in biological systems communicate in a type of "chemical braille", where molecular-scale bumps and grooves of one protein molecule is read by the physical interaction of another protein. Detailed understanding of the molecular-level communications of these protein-protein interactions is necessary to determine the fundamental rules governing both normal and abnormal cellular function. The ability to both characterize and manipulate these interactions at the molecular level has proven challenging. The Pomerantz laboratory at the University of Minnesota works to understanding the chemical details of protein-protein interactions, with the long-term goal of developing ways to stop protein-protein communications that result in harmful cellular functions. To achieve this goal, novel fluorine-based "probe" protein molecules report on difficult-to-detect protein-protein interactions due to their intrinsic responsiveness in differing chemical environments. Research efforts are integrated with educational activities through course-based undergraduate research experiences (CUREs) designed to increase student success and increase retention in science-technology-engineering-mathematics career paths. Broadened impact of the research project occurs through collaborations with Gustavus Adolphus College, as well as an educational bootcamp that expands undergraduate student exposure to chemical biology research and mentoring by the Pomerantz team.The Chemistry of Life Process Program in the Division of Chemistry at the NSF is funding Dr. William Pomerantz from the University of Minnesota- Twin Cities for development of fluorine nuclear magnetic resonance spectroscopy (NMR) methods for detecting, quantifying, and defining novel modes of interactions at transcription factor protein-protein interfaces, including those that involve epigenetic regulatory proteins. The long-term research goals of the Pomerantz team are to develop chemical tools to probe the biological roles of transcription factor protein interactions with specific interest in epigenetic gene regulation. This research combines fluorine-19 NMR approaches developed in the Pomerantz lab with additional biophysical methods to characterize native and synthetic ligand binding interactions. This research provides a detailed understanding of the role of aromatic amino acids involved in the protein allosteric network for regulating the KIX protein-protein interaction domain of CREB binding protein (CBP). A recent discovery of the Pomerantz team is the identification of a new protein interaction site on the KIX domain targeted by synthetic small molecules. A key fundamental question to be addressed is the nature of the interaction for other proteins with the newly discovered small-molecule binding site. The Pomerantz team structurally characterizes this site by NMR and uses photo-crosslinking and proteomic methods to evaluate if other proteins occupy this binding site for allosterically regulating KIX function. They evaluate potential domain-domain interactions when studied first as separate domains and ultimately the full-length construct. The research aims are integrated with an educational objective of creating course-based undergraduate research experiences, which result from authentic undergraduate chemistry student research experiences afforded by the study of protein-ligand interactions with fluorine-19 NMR methods.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.