Quantitative Description of Phosphorylation Effects on Disordered Protein Structure

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 DESCRIPTION (provided by applicant): We have developed a method for characterizing intrinsically disordered protein (IDP) structures that we will use to establish a predictive and quantitative model that describes protein phosphorylation in disordered regions. Protein phosphorylation is used biologically as a mechanism to control many critical life processes and phosphorylation usually occurs within protein regions that are intrinsically disordered. Accordingly, quantitative descriptions and molecular models of phosphorylation effects on disordered protein structures are needed to understand the molecular basis of key aspects of development, aging, and disease. The method that we have developed to study IDPs is useful because it can analyze structural relationships in detail and with quantitative precision, linking microscopic residue-specific descriptors, such as intrinsic conformational propensities, to macroscopic global metrics like the hydrodynamic radius (Rh). For example, we demonstrated that the effects of glycine substitutions on Rh could be used to estimate per-residue polyproline II (PPII) propensities in disordered proteins. Our results also provided evidence that PPII propensities and charge effects on IDP structures are linked, indicating a possible correlation between PPII structure, which is a dominant conformation in disordered proteins, and phosphorylation effects, which are key regulators of IDP activity. For the studies that are proposed in this R-15 application, we will use the intrinsically disordered N-terminal region of th p53 tumor suppressor protein consisting of residues 1-93 as our experimental model system. Aberrant p53 activity has been associated with numerous human cancers. The objectives of this application are to test the ability of our method to extract structural detail from the p53(1-93) system, measure intrinsic PPII propensities for the common amino acid types, establish if intrinsic PPII propensities depend on nearest neighbor sequence details, and apply our technology for investigating phosphorylation mechanisms quantitatively by modeling the effects of charge and PPII propensities on IDP structure for comparison to experiments that will measure charge, phosphorylation, and PPII effects on Rh. The data measured will be fundamental for establishing a new and predictive approach for characterizing IDP structures, investigating their biological roles, and modeling how their biological activities are regulated. The goals of this application are part of our long-term objective of developing quantitative models of IDP structure/function relationships. The data from this study will advance IDP molecular biophysics by: 1) developing a method for measuring PPII propensities in disordered proteins, 2) establishing if local sequence details influence intrinsic PPII propensities, 3) quantifying coupling between charge and PPII effects on IDP structure, and 4) measuring quantitatively the effects of phosphorylation on IDP structure.