Probing the Folding Mechanism of Intrinsically Disordered Proteins: The pKID/KIX

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DESCRIPTION (provided by applicant): Within the protein folding field, intrinsically disordered proteins (IDPs) are emerging as an important yet often overlooked class of molecules. These proteins, which are disordered under physiological conditions, have been found to make up as much as 30% of the human proteome, and are often cell signaling molecules or DNA binding proteins. Understanding the overall mechanism of action for these proteins will be very important in efforts to improve upon cell signaling. IDPs are found to fold upon association with either a membrane, small molecule, or another protein. Though individual IDPs can fold in different ways, i.e. by binding a ligand first and then folding or vice versa, determining these details for specific IDPs will be of the utmost importance for trying to stabilize their complex and fashion pharmaceutical drugs to control their action. This proposal aims to study the pKID/KIX complex, which are two domains isolated from the transcription factor CREB and its activator CREB binding protein (CBP). These two domains are where CREB (via pKID, the IDP) and CBP (via KIX, the ligand) associate, and only upon CBP binding to it can CREB actively promote downstream signaling. Studies have long shown that CREB-dependent signaling is important for memory development and brain aging, and even suggested that this signaling may be impaired by Alzheimer's. Therefore, to increase understanding of the association between CREB and CBP in the hope of ultimately stabilizing CREB/CBP association and developing drugs to promote continued CREB-dependent signaling in the aging populace, this proposal aims to study the folding and binding of pKID to KIX to determine molecular details of the complex formation. Work will be done using FRET and infrared-based kinetic experiments to understand the detailed structural and kinetic order of pKID folding and binding to KIX. Additional experiments will show how important individual residues within pKID are for folding, and how increasing the stability of pKID affects its binding with KIX. The results of these experiments will provide insight into how IDPs work as a whole, as well as determine important molecular characteristics of CREB/CBP binding, which can then be used to artificially improve their association and possibly forestall aging.