Biophysical characterization of protein-protein interactions involving intrinsically disordered proteins

Intrinsically disordered proteins and regions (IDPs/Rs) are proteins that do not form stable and well-defined structures in their free states but rather occupy an ensemble of conformations that change over time while still staying functional. They are prevalent in the eukaryotic proteome and are involved in various vital processes in the cell where they often interact with their binding partners through coupled binding and folding reactions. The knowledge on the molecular details of these interactions is still limited as is the role of dynamics and conformational entropy changes. In this thesis binding interactions between IDPs and a folded protein domain have been studied in more detail. The rate-limiting transition states (TS) of binding have been examined using kinetic experiments and protein engineering (F-value analysis), and the picosecond to nanosecond backbone and side-chain dynamics of these interactions have been studied with nuclear magnetic resonance (NMR) spectroscopy. To study these properties the globular TAZ1 domain of the CREB binding protein (CBP) and three of its interaction partners, the disordered transactivation domains of STAT2, HIF-1a and RelA have been selected. At the rate limiting transition states of binding for TAZ1/TAD-STAT2 and TAZ1/CTAD-HIF-1a native hydrophobic binding contacts are largely absent. These interactions are instead formed cooperatively after passing the rate-limiting barrier. The results from the backbone and side-chain dynamic studies show that the internal motions for both binding partners are significantly affected by the interactions. Changes in dynamics upon binding correspond to conformational entropy changes that contribute significantly to the binding thermodynamics, and are in the same order of magnitude as the binding enthalpy. Additionally, the conformational entropy changes for TAZ1 vary when binding to the different IDPs, demonstrating the importance of conformational entropy. In conclusion, this work contributes to the understanding of the nature of binding interactions involving intrinsically disordered proteins