Structural Effects of Protein Phosphorylation and O-GlcNAcylation

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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.