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通过一种“化学盲文”进行交流，其中一种蛋白质分子的分子尺度的凸起和凹槽被另一种蛋白质的物理相互作用读出。为了确定控制正常和异常细胞功能的基本规则，有必要详细了解这些蛋白质-蛋白质相互作用的分子水平通信。事实证明，在分子水平上描述和操纵这些相互作用的能力是具有挑战性的。明尼苏达大学的波美兰茨实验室致力于了解蛋白质-蛋白质相互作用的化学细节，长期目标是开发方法来阻止导致有害细胞功能的蛋白质-蛋白质通讯。为了实现这一目标，新型的基于氟的“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。研究工作与教育活动相结合，通过以课程为基础的本科生研究经验(CURE)，旨在提高学生的成功并增加在科学-技术-工程-数学职业道路上的留存。该研究项目的广泛影响是通过与Gustavus Adolphus学院的合作以及一个教育训练营来实现的，该训练营扩大了本科生接触化学生物学研究的机会，并得到了Pmerantz团队的指导。NSF化学部的生命过程化学项目正在资助明尼苏达大学双子城大学的William Pmerantz博士开发氟核磁共振(NMR)方法，用于检测、量化和定义转录因子蛋白质-蛋白质界面上的新的相互作用模式，包括涉及表观遗传调节蛋白质的方法。波美兰茨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质相互作用的生物学作用，并对表观基因调控产生特殊兴趣。这项研究将波美兰茨实验室开发的氟-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作用。本研究详细了解了芳香族氨基酸在蛋白变构网络中对CREB结合蛋白(CBP)的KIX蛋白相互作用结构域的调控作用。波美兰茨团队最近的一项发现是在KIX结构域上发现了一个新的蛋白质相互作用位点，以合成小分子为靶标。一个需要解决的关键基本问题是其他蛋白质与新发现的小分子结合部位的相互作用的性质。Pmerantz研究小组通过核磁共振对该结合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了该结合位点，以变构方式调节KIX功能。当首先作为单独的结构域进行研究，并最终作为全长结构进行研究时，他们评估潜在的结构域-结构域相互作用。这项研究的目的是与一个教育目标相结合，创造基于课程的本科生研究经验，这些经验来自于用氟-19核磁共振方法研究蛋白质-配体相互作用所提供的真实的本科生研究经验。这个奖项反映了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。

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.