Structural Effects of Protein Phosphorylation and O-GlcNAcylation

蛋白质磷酸化和O-GlcNacyl化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，也少于大多数植物。真核生物(从酵母到植物再到人类)生命的复杂性在一定程度上是由于蛋白质动态发生的修饰，由于一系列潜在的修饰，蛋白质在不同的时间具有不同的功能。细胞内蛋白质最常见的修饰包括磷酸化，即一个或多个磷酸基团的添加，以及O-GlcN酰化，即添加糖。这些修饰是广泛的细胞功能的核心。磷酸化和O-GlcN酰化都可以发生在蛋白质内的同一位置，但这些修饰如何影响这些蛋白质的结构，从而如何影响这些蛋白质的功能，通常还知之甚少。这项工作将提供一个全面的理解如何磷酸化和O-GlcN酰化影响蛋白质结构。这项工作将培训本科生、研究生和博士后研究员掌握21世纪科学进步所必需的多学科方法，使他们成为能够在不同领域工作的科学领导者。本科生实验室教育中的一个重大挑战是让学生参与当前的研究。这项工作还将开发新的本科实验室，既要对学生进行多种技术的综合培训，又要让学生直接参与科学研究。这些本科实验室实验的结果将作为科学文献的一部分，并将为教授学生结构生物学的方法提供新的方法。蛋白质磷酸化和O-GlcN酰化是所有真核生物信号转导的核心。蛋白质磷酸化和O-GlcN酰化是相互竞争的细胞内蛋白质丝氨酸和苏氨酸残基的翻译后修饰，调节信号转导通路以控制细胞功能。磷酸化和O-GlcN酰化具有不同的功能效应，它们在功能上有时互补，有时对立。这项工作将发展新的原理和方向来理解生物功能和具体理解蛋白质磷酸化和O-GlcNacyl化的结构效应。这项工作将结合理论、生物信息学、溶液实验和X射线结晶学实验，为理解翻译后修饰对结构和功能的影响提供详细的框架，并应用于从酵母到植物再到哺乳动物的系统。这项研究将使用多肽和蛋白质，结合圆二色谱、核磁共振光谱、X射线结晶学和从头计算的结构分析，独立和比较地考察磷酸化和O-GlcN酰化对调节蛋白质结构的影响，特别是考察丝氨酸和苏氨酸残基修饰的差异。合成的多肽将分析不同环境下丝氨酸和苏氨酸的磷酸化和O-GlcN酰化对结构的影响，包括简短的模型多肽，适合详细的核磁共振分析、从头计算和小分子X射线结晶学，以及较大的多肽和蛋白质，适合热力学表征、酶学和蛋白质X射线结晶学。这些实验将导致对磷酸化和O-GlcN酰化对蛋白质结构和功能的翻译后修饰在细胞内信号转导中的作用以及蛋白质中丝氨酸和苏氨酸残基的不同作用的新的见解。这项工作还将发展对磷酸化和O-GlcN酰化介导的结构变化基础的新的理论理解，适用于更准确地对蛋白质进行翻译后修饰的建模。这项工作将专门开发新的以发现为导向的本科实验室，培训学生固相反应、肽合成、氨基酸的比较研究以及使用一维和二维核磁共振进行结构分析，同时让学生在入门实验室中为科学知识的发展做出贡献。

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.