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

激酶是改变细胞中特定化学反应速率的生物分子。它们和这些化学反应的产物代表了生命所有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之外，特定激酶对特定分子产物的效率是将输入信号靶向不同生理输出的关键。该项目的目标是了解这种调节的性质，在空间和时间上具有足够的分辨率，以提供干扰和最终设计信号网络的方法，这些信号网络将特定的输入信号驱动到定义的细胞反应中。该项目还将为一名研究生和几名本科生研究人员提供一个肥沃的培训基地，涵盖计算方法学，实验NMR和酶生物化学等各种技能。本科生将从从纽约（CUNY）七所社区学院之一转入纽约（CCNY）的学生中挑选，目的是鼓励他们在STEM学科中接受高等教育。值得注意的是，这个项目的研究生以前是一名转学生，他选择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代表细胞内信号转导的中心机制，并且在几乎所有生理过程中起关键作用。这种转移是由称为蛋白激酶的生物催化剂实现的，并且涉及激酶、底物和ATP之间的化学前复合物的形成。最近的研究表明，在这种预化学复合物的背景下，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，并有助于其特异性激酶/底物对之间的变异性。激酶/底物对之间的磷酸化效率的这些变化对于维持细胞中同时发生的许多基于磷酸化的信号传导事件的精细调谐网络是必要的。理解这些效率差异需要表征构成预化学复合物的构象状态的原子细节。这个项目，使用尖端的计算方法，溶液状态核磁共振（NMR）方法和生化分析相结合，旨在表征的构象状态，定义的预化学复杂的目标，解决以下问题：是什么决定了磷酸化的效率内激酶底物ATP预化学复杂？为该项目选择的模型系统涉及促分裂原活化蛋白激酶ERK 2和来自其天然底物的肽，转录因子Elk-1，它们以不同的效率磷酸化。该奖项反映了NSF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。

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.