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

碳检测核磁共振研究内在无序的蛋白质翻译后修饰对DNA中编码的遗传信息的访问必须既快速又严格控制，以便细胞能够容易地适应其环境，或对生长信号做出快速反应。一种调节机制涉及使用具有高度灵活区域的蛋白质，这些区域可以根据信号进行化学修饰。这些化学修饰影响这些蛋白质与DNA的结合或访问，从而决定遗传信息是可访问和表达的，还是隐藏和抑制的。因此，这些高度灵活的蛋白质的化学修饰可以对细胞的反应施加及时的控制，在活跃和不活跃状态之间切换目标，并允许细胞快速调动目前所需的资源。目前的技术很难观察到高度灵活的区域；这一事实极大地限制了我们对它们如何发挥作用的理解。研究人员将突破当前技术的界限，探索控制遗传信息表达的高度灵活蛋白质的化学转换的结构后果。这个项目解决了我们在基因表达是如何调控的知识方面的一个重大空白。这项研究将培训不同教育水平的初级科学家，利用物理学的原理和定量规律，寻求对生物过程生物化学的基本见解。在其他外展工作中，调查员将把来自贫困背景的中学生带到宾夕法尼亚州立大学，展示科学和工程领域的高等教育带来的机会。为了实现这个项目的目标，将实施新的实验方法来研究高度柔韧的蛋白质的核磁共振波谱。虽然质子相对容易通过核磁共振观察，但PI表明，相对更具挑战性的直接碳检测产生了更多定量和完整的内在无序多肽信息。最近，PI使用这项技术来证明RNA聚合酶II的丝氨酸磷酸化如何提供一个结构开关，在调节这个至关重要的酶方面发挥作用。等电点现在将扩大高度灵活的蛋白质的多样性，这些蛋白质可以用碳检测核磁共振进行研究。具体地说，将开发克服目前芳香氨基酸酪氨酸和苯丙氨酸盲点的方法。其次，该项目将扩展这些研究，以解决苏氨酸磷酸化的结构性后果。最后，响应细胞和环境信号而获得磷酸盐的蛋白质通常也会获得甲基。这些修改可以施加组合控制，通常是在竞争中。因此，将进行实验来检验这样的假设，即在氨基酸赖氨酸上添加甲基也会诱导结构转变，类似于PI观察到的与磷酸盐掺入相关的结构转变。因此，这项研究和相关的培训活动将产生对基因调控的基本的、分子水平的见解。这一奖项反映了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。

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.