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Carbon-Detected NMR Studies of Intrinsically Disordered Protein Post-Translational Modification

本质无序蛋白质翻译后修饰的碳检测核磁共振研究

基本信息

批准号：
1932730
负责人：
Scott Showalter
金额：
$ 95.88万
依托单位：
Pennsylvania State Univ University Park
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1932730&HistoricalAwards=false
关键词：
Carbon Detected NMR Studies Intrinsically

项目摘要

Carbon-Detected NMR Studies of Intrinsically Disordered Protein Post-Translational ModificationAccess to the genetic information encoded in DNA must be both rapid and under tight control so that a cell can readily adapt to its environment, or rapidly respond to growth signals. One regulatory mechanism involves the use of proteins that have highly flexible regions that can be chemically modified in response to signals. These chemical modifications impact how these proteins associate with, or access, DNA and thus determine whether genetic information is accessible and expressed or hidden and suppressed. Thus, chemical modifications of these highly flexible proteins can exert timely control over cellular responses, acting to toggle their targets between active and inactive states and allowing the cell to quickly mobilize the resources it requires at the moment. Highly flexible regions are difficult to observe with current technologies; this fact significantly limits our understanding of how they function. The investigator will push the boundaries of current technologies and explore the structural consequences of chemical toggles of highly flexible proteins that control the expression of genetic information. This program addresses a significant gap in our knowledge of how gene expression is regulated. The research will train junior scientists at multiple education levels to seek fundamental insight into the biochemistry of biological processes using the principles and quantitative laws from the physical sciences. Among other outreach efforts, the investigator will bring middle school students from underprivileged backgrounds to Penn State to demonstrate the opportunities that arise from higher education in science and engineering fields. To achieve this project's objectives, new experimental methodology will be implemented for nuclear magnetic resonance (NMR) spectroscopy of highly flexible proteins. While protons are relatively easy to observe by NMR, the PI has shown that the comparatively more challenging direct-detection of carbon yields more quantitative and complete information for intrinsically disordered polypeptides. Recently, the PI used this technique to demonstrate how serine phosphorylation of RNA polymerase II provides a structural switch that plays a role in regulating the critically important enzyme. The PI will now broaden the diversity of the highly flexible proteins that can be studied with carbon-detected NMR. Specifically, methods will be developed to overcome a current blind-spot for the aromatic amino acids tyrosine and phenylalanine. Second, the project will extend these studies to address the structural consequences of threonine phosphorylation. Finally, proteins that acquire phosphate in response to cellular and environmental signals often also acquire methyl groups. These modifications can exert combinatorial control, often in competition. Therefore, experiments will be performed to test the hypothesis that addition of methyl groups to the amino acid lysine also induces structural transitions, akin to those the PI has observed in association with phosphate incorporation. As a result, this research and associated training activities will yield fundamental, molecular level insights into gene regulation.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.

利用碳探测核磁共振技术研究蛋白质内无序的翻译后修饰对DNA中编码的遗传信息的获取必须既快速又严格控制，这样细胞才能容易地适应其环境，或对生长信号迅速作出反应。一种调节机制涉及使用具有高度灵活区域的蛋白质，这些区域可以响应信号进行化学修饰。这些化学修饰影响这些蛋白质如何与DNA结合或接触，从而决定遗传信息是可接触和表达还是隐藏和抑制。因此，这些高度灵活的蛋白质的化学修饰可以及时控制细胞反应，在活性和非活性状态之间切换它们的目标，并允许细胞快速动员它目前所需的资源。高度灵活的区域很难用现有技术观察到;这一事实大大限制了我们对它们如何运作的理解。研究人员将推动当前技术的边界，并探索控制遗传信息表达的高度灵活的蛋白质的化学切换的结构后果。该计划解决了我们对基因表达如何调控的知识中的一个重大空白。该研究将培养多个教育层次的初级科学家，利用物理科学的原理和定量定律，寻求对生物过程生物化学的基本见解。在其他外联工作中，调查人员将把来自贫困背景的中学生带到宾夕法尼亚州立大学，以展示科学和工程领域高等教育带来的机会。为了实现该项目的目标，将实施新的实验方法，用于高柔性蛋白质的核磁共振（NMR）光谱。虽然质子相对容易通过NMR观察，但PI已经表明，相对更具挑战性的碳直接检测可以为本质无序的多肽提供更定量和完整的信息。最近，PI使用这种技术来证明RNA聚合酶II的丝氨酸磷酸化如何提供在调节至关重要的酶中起作用的结构开关。PI现在将扩大可以用碳检测NMR研究的高度灵活的蛋白质的多样性。具体而言，将开发方法来克服目前的盲点的芳香族氨基酸酪氨酸和苯丙氨酸。其次，该项目将扩展这些研究，以解决苏氨酸磷酸化的结构后果。最后，响应细胞和环境信号而获得磷酸盐的蛋白质通常也获得甲基。这些修改可以发挥组合控制，往往在竞争中。因此，将进行实验以检验向氨基酸赖氨酸添加甲基也诱导结构转变的假设，类似于PI在磷酸盐掺入中观察到的结构转变。因此，这项研究和相关的培训活动将产生基本的，分子水平的见解基因regulation.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。