EAGER: Nature of the Pre-chemistry Ensemble in Protein Kinases

激酶是一种生物分子，可以改变细胞中特定化学反应的速度。它们和这些化学反应的产物代表着生命各个领域细胞内信号网络的中心枢纽。除了亚细胞定位和时间表达，特定的激酶对特定的分子产物的效率是将输入信号定向到不同的生理输出的关键。该项目的目标是在空间和时间上以足够的分辨率理解这一规定的性质，以提供干扰并最终设计驱动特定输入信号向定义的细胞响应的信令网络的手段。该项目还将为一名研究生和几名本科生提供一个肥沃的培训场地，掌握从计算方法、实验核磁共振和酶生物化学在内的各种技能。这些本科生将从纽约城市大学(CUNY)所属的七所社区学院中的一所转学到纽约城市学院(CCNY)，目的是鼓励他们在STEM学科上接受高等教育。值得注意的是，在这个项目上预期的研究生以前是一名转校生，他选择了攻读生物化学博士学位。磷酸基团从三磷酸腺苷(ATP)转移到底物蛋白的特定丝氨酸、苏氨酸或酪氨酸残基上是细胞内信号转导的中心机制，在几乎所有的生理过程中都起着关键作用。这种转移是由被称为蛋白激酶的生物催化剂实现的，涉及到在激酶、底物和ATP之间形成化学前复合体。最近的研究表明，在这种前化学复合体的背景下，激酶的催化元件和底物的磷酸受体区域的结构动力学的性质决定了磷酸转移反应的效率，并导致了特定的激酶/底物对之间的差异。激酶/底物对之间的这些磷酸化效率的差异对于维持细胞中同时发生的许多基于磷酸化的信号事件的微调网络是必要的。要了解这些效率上的差异，就需要在原子细节上表征组成前化学络合物的构象状态。该项目结合使用尖端计算方法、溶液状态核磁共振方法和生化分析方法，旨在表征定义化学前复合体的构象状态，以解决以下问题：是什么决定了激酶-底物-ATP化学前复合体中的磷酸化效率？为该项目选择的模型系统涉及丝裂原激活蛋白激酶ERK2和来自其天然底物转录因子ELK-1的多肽，它们以不同的效率被磷酸化。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。

Kinases are biological molecules that modify the rate of specific chemical reactions in cells. They and the products of these chemical reactions represent central hubs in the intracellular signaling networks in all domains of life. In addition to sub-cellular localization and temporal expression, the efficiencies of particular kinases towards specific molecular products is key in targeting input signals towards distinct physiological outputs. The goal of this project is to understand the nature of this regulation with sufficient resolution in space and time to provide the means to perturb and ultimately design signaling networks that drive specific input signals towards defined cellular responses. This project will also provide a fertile training ground for a graduate student and several undergraduate researchers in a wide variety of skills that range from computational methodology, experimental NMR and enzyme biochemistry. The undergraduates will be drawn from the pool of students who transfer from one of the seven community colleges that are part of the City University of New York (CUNY) into the City College of New York (CCNY) with a goal of encouraging them to pursue higher education in the STEM disciplines. It is notable that the graduate student who is expected on this project was formerly a transfer student who has chosen to pursue his PhD degree in Biochemistry. The transfer of a phosphate group from adenosine triphosphate (ATP) to specific serine, threonine or tyrosine residues of substrate proteins represents a central mechanism of signal transduction within cells and plays a critical role in almost all physiological processes. This transfer is enabled by biocatalysts known as protein kinases and involves the formation of a pre-chemistry complex between the kinase, the substrate and ATP. Recent studies suggest that the nature of the structural dynamics of the catalytic elements of the kinase and the phospho-acceptor region of the substrate in the context of this pre-chemistry complex determines the efficiency of the phospho-transfer reaction and contributes to its variability between specific kinase/substrate pairs. These variations in phosphorylation efficiency between kinase/substrate pairs is necessary to maintain the finely tuned network of numerous phosphorylation based signaling events occurring simultaneously in the cell. An understanding of these differences in efficiency requires the characterization in atomic detail of the conformational state/states that comprise the pre-chemistry complex. This project, using a combination of cutting-edge computational approaches, solution-state nuclear magnetic resonance (NMR) methods and biochemical assays, aims to characterize the conformational states that define the pre-chemistry complex towards the goal of addressing the following question: what determines the efficiency of phosphorylation within a kinase-substrate-ATP pre-chemistry complex? The model system chosen for this project involves the mitogen-activated protein kinase ERK2 and peptides derived from its natural substrate, the transcription factor Elk-1, that are phosphorylated with differing efficiencies.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.