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Structural Effects of Protein Phosphorylation and O-GlcNAcylation

蛋白质磷酸化和 O-GlcNAc 酰化的结构效应

基本信息

批准号：
1616490
负责人：
Neal Zondlo
金额：
$ 69.93万
依托单位：
University of Delaware
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616490&HistoricalAwards=false
关键词：
Structural Effects Protein Phosphorylation GlcNAcylation

项目摘要

TITLE: Structural Effects of Protein Phosphorylation and O-GlcNAcylationThe human genome encodes for approximately 22,000 proteins, a relatively small number comparable to that in worms and less than that in most plants. The complexity of life in eukaryotic organisms (from yeast to plants to humans) is due in part to modifications that occur dynamically to proteins, which allow proteins to have different functions at different times as a result of a series of potential modifications. The most common modifications of proteins within cells include phosphorylation, the addition of one or more phosphate groups, and O-GlcNAcylation, the addition of a sugar. These modifications are central to a wide range of cellular functions. Both phosphorylation and O-GlcNAcylation can occur on the same sites within proteins, but how these modifications affect the structure of these proteins, and therefore how they can affect the function of these proteins, is generally poorly understood. This work will provide a comprehensive understanding of how phosphorylation and O-GlcNAcylation affect protein structure. This work will train undergraduate researchers, graduate students, and post-doctoral fellows in multidisciplinary methods in science that are necessary for scientific advances in the 21st century, preparing them to be scientific leaders who can work across diverse fields. One significant challenge in undergraduate laboratory education is the engagement of students in current research. This work will also develop new undergraduate laboratories that both train students comprehensively in multiple techniques and that directly involve students in scientific research. The results of these undergraduate laboratory experiments will both be included as part of the scientific literature and will provide new methods for teaching students in methods in structural biology.Protein phosphorylation and O-GlcNAcylation are central to signal transduction in all eukaryotes. Protein phosphorylation and O-GlcNAcylation are competing intracellular protein post-translational modifications of serine and threonine residues, which modulate signal transduction cascades to control cellular function. Phosphorylation and O-GlcNAcylation have diverse functional effects, which are sometimes complementary and sometimes opposing in function. This work will develop new principles and directions to understand biological function and to specifically understand structural effects of protein phosphorylation and O-GlcNAcylation. This work will combine theory, bioinformatics, experiments in solution, and experiments by x-ray crystallography to provide a detailed framework for understanding the effects of post-translational modifications on structure and function, with application to systems from yeast to plants to mammals. The research will use peptides and proteins combined with structural analysis by circular dichroism, NMR spectroscopy, x-ray crystallography, and ab initio calculations to examine independently and comparatively the effects of phosphorylation and O-GlcNAcylation on modulating the structure of proteins, specifically examining the differences due to modification at serine versus threonine residues. Peptides will be synthesized to analyze the effects of phosphorylation and O-GlcNAcylation of serine and threonine on structure in diverse contexts, including in short model peptides amenable to detailed NMR analysis, ab initio calculations, and small-molecule x-ray crystallography, and in larger peptides and proteins amenable to thermodynamic characterization, enzymology, and protein x-ray crystallography. These experiments will lead to new insights in the roles of the post-translational modifications of phosphorylation and O-GlcNAcylation on protein structure and function in intracellular signaling and on the differential roles of serine and threonine residues in proteins. This work will also develop new theoretical understanding of the bases for phosphorylation- and O-GlcNAcylation-mediated structural changes, applicable to more accurate modeling of proteins with post-translational modifications. This work will specifically develop new discovery-oriented undergraduate laboratories that will train students in solid-phase reactions, peptide synthesis, comparative studies of amino acids, and structural analysis using 1-D and 2-D NMR, while simultaneously having students in introductory labs contribute to the development of scientific knowledge.

标题：蛋白质磷酸化和O-GlcNAcylation的结构效应人类基因组编码大约22，000种蛋白质，与蠕虫相比相对较少，并且少于大多数植物。真核生物（从酵母到植物再到人类）生命的复杂性部分是由于蛋白质动态发生的修饰，这使得蛋白质在不同时间具有不同的功能，这是一系列潜在修饰的结果。细胞内蛋白质最常见的修饰包括磷酸化，添加一个或多个磷酸基团，以及O-GlcNAc酰化，添加糖。这些修饰对广泛的细胞功能至关重要。磷酸化和O-GlcNAc化都可以发生在蛋白质内的相同位点上，但是这些修饰如何影响这些蛋白质的结构，因此它们如何影响这些蛋白质的功能，通常知之甚少。这项工作将提供一个全面的了解如何磷酸化和O-GlcNAcylation影响蛋白质结构。这项工作将培养本科生研究人员，研究生和博士后研究员在科学的多学科方法，是必要的科学进步在21世纪世纪，准备他们成为科学领导人谁可以跨不同领域的工作。本科实验室教育的一个重大挑战是学生参与当前的研究。这项工作还将开发新的本科生实验室，既全面培训学生多种技术，又直接让学生参与科学研究。这些本科生实验室实验的结果将被列入科学文献的一部分，并将提供新的方法，为教学学生在结构biology.Protein磷酸化和O-GlcNAcylation的方法是中央在所有真核生物的信号转导。蛋白质磷酸化和O-GlcNAc化是丝氨酸和苏氨酸残基的竞争性细胞内蛋白质翻译后修饰，其调节信号转导级联以控制细胞功能。磷酸化和O-GlcNAc化具有不同的功能效应，它们在功能上有时是互补的，有时是相反的。这项工作将开发新的原则和方向，以了解生物功能，并具体了解蛋白质磷酸化和O-GlcNAc酰化的结构效应。这项工作将结合联合收割机理论，生物信息学，实验解决方案，并通过X射线晶体学实验，以提供一个详细的框架，了解翻译后修饰的结构和功能的影响，从酵母到植物到哺乳动物的系统的应用。该研究将使用肽和蛋白质结合圆二色谱，NMR光谱，X射线晶体学和从头计算的结构分析，以独立和比较地研究磷酸化和O-GlcNAc化对调节蛋白质结构的影响，特别是研究由于丝氨酸与苏氨酸残基修饰的差异。将合成肽，以分析丝氨酸和苏氨酸的磷酸化和O-GlcNAc化对不同背景下结构的影响，包括适合详细NMR分析，从头计算和小分子X射线晶体学的短模型肽，以及适合热力学表征，酶学和蛋白质X射线晶体学的较大肽和蛋白质。这些实验将导致新的见解的作用的翻译后修饰的磷酸化和O-GlcNAcylation蛋白质的结构和功能的细胞内信号和蛋白质中的丝氨酸和苏氨酸残基的差异作用。这项工作还将发展新的理论理解的基础磷酸化和O-GlcNAc酰化介导的结构变化，适用于更准确的蛋白质与翻译后修饰的建模。这项工作将专门开发新的以发现为导向的本科实验室，这些实验室将培养学生进行固相反应，肽合成，氨基酸比较研究以及使用1-D和2-D NMR进行结构分析，同时让学生在入门实验室中为科学知识的发展做出贡献。