PROJECT SUMMARY Intrinsically disordered protein regions (IDRs) are critical regulatory modules for many processes in the eukaryotic cell, such as transcription regulation and cell cycle control. Many diseases are characterized by dysregulation of the proteins involved in these processes, including neurodegenerative diseases and cancers. Post-translational modifications (PTMs), like phosphorylation, within IDRs can control their conformational states and intermolecular interactions toward a specific functional outcome; as such, a molecular-level understanding of the effects of phosphorylation and other PTMs on IDR behavior will advance the development of therapeutic strategies targeted to these regulatory proteins and their modifications. The project proposed herein centers on the C-terminal IDR of the Retinoblastoma protein (Rb) and its multi-site phosphorylation associated with cell cycle progression. Rb functions and their dysregulation in cancers have been studied extensively, but the specific mechanistic consequences of phosphorylation on the C-terminal IDR (CTD) that support Rb control of the cell cycle are not clear. Therefore, this study will test the hypothesis that Rb-CTD undergoes both global and local conformational changes associated with multi-site phosphorylation. This project leverages tandem computational and experimental approaches to overcome technical challenges associated with the structural characterization of IDRs. Specifically, all-atom simulations and solution-state biophysical measurements will reveal the atomistic details of Rb IDR behavior with and without multi-site phosphorylation. Aim 1 will expand on the existing infrastructure for molecular simulations of IDRs by development and incorporation of parameters for phosphoserine and phosphothreonine. Aim 2 will test the hypothesis that Rb phosphorylation confers functional switching by affecting the IDR conformations presented to putative binding partners. A strategic combination of all-atom simulations, NMR spectroscopy, and single-molecule fluorescence will provide quantitative details about the conformations preferred by unmodified or phosphorylated Rb-CTD. Finally, Aim 3 will interrogate a known interaction between phosphorylated Rb-CTD and an adjacent Rb folded domain. In this aim, small-angle X-ray scattering and NMR spectroscopy experiments will be used to test the hypothesis that electrostatic forces introduced through phosphorylation drive the intramolecular interaction of Rb in cis. Together, the research and training objectives outlined in this proposal strongly align with the mission of the NIH to understand the mechanisms of disease and to foster the future generation of academic investigators in biomedical science. Further, the completion of these studies will provide valuable insights into the basic biophysical underpinnings of Rb- and other IDR-linked diseases.