Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Complex dynamic behavior dictates many key aspects of protein function. Intrinsically disordered proteins (IDPs), which are involved in cancer and neurodegenerative diseases, exhibit conformational changes on nanosecond to millisecond timescales which are essential for their regulatory mechanisms. Despite an intense multidisciplinary focus on IDPs in recent years, a reasonable understanding of the physical mechanisms underlying their structural disorder and the dynamic nature of their interactions is still lacking.
Sic1 is a cyclin-dependent kinase inhibitor involved in the regulation of the yeast cell cycle, which upon multi-site phosphorylation binds to a single site on its acceptor protein, Cdc4. NMR studies indicated the presence of a dynamic complex of Sic1:Cdc4, with Sic1 remaining predominantly disordered upon phosphorylation and binding. The physical basis for this dynamic, multivalent binding of Sic1 to Cdc4 is unknown, although electrostatic interactions are thought to play an important role. In the nervous system, the cap-dependent translation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. One of those proteins, 4E-BP2, acts like a regulatory switch: in the non-phosphorylated state is largely disordered and bound to eIF4E, while in the multi-phosphorylated state it folds and detaches, allowing another protein (eIF4G) to dock onto eIF4E and initiate translation. The main question for this system concerns the role of phosphate groups, whether they stabilize the folding of 4E-BP2, or destabilize the interaction with eIF4E, or both.
We propose a research program focused on studying the conformational states and the dynamics of fluorescently labelled Sic1 and 4E-BP2 using single-molecule fluorescence techniques, such as single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Correlation Spectroscopy (FCS). Our previous smFRET studies point to a rather shallow, but rugged energy landscape of IDPs and to electrostatics modulating their compaction and conformational dynamics. Building on our recent progress, polymer physics models and computational modelling will be used to infer global dimensions of IDPs from smFRET data. Our main goals are to quantify the distribution of conformations, local and global dynamics, binding kinetics and energetics for two paradigmatic IDPs, at first in vitro and then in live cells. The results will help change the classic structure-function paradigm in biology to more dynamic alternatives, will provide insight into the variety of creative regulatory mechanisms provided by IDPs, and will be an important step towards designing synthetic or biologic drugs against their aberrant activities.