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.