Assessing the Structural Ensemble and Folding Propensity of Intrinsically Disordered Proteins

The coupled folding and binding mechanism of intrinsically disordered proteins (IDPs) creates energetic consequences that yield strong, specific interactions yet with the ability to adapt toward multiple different binding targets. To understand the role of IDP structural flexibility, characterization of the conformational ensemble of the unbound molecule along with determination of its trajectory toward the bound state is required. We used small-angle neutron scattering (SANS) to investigate the structural ensemble of an intrinsically disordered region of activator for thyroid hormone and retinoid receptor (ACTR), a binding partner of the transcription coactivator CREB binding protein (CBP). The structures generated computationally using the ensemble optimization method (EOM) to fit the SANS data displayed size distributions that indicate unbound ACTR samples compact states that are comparable to the bound form and may be most amenable toward binding CBP. We also investigated the folding propensity of ACTR and its target IDP region of CBP using circular dichroism spectroscopy and osmotic stress. Both IDPs adopt increased alpha-helical content with applied osmotic stress for several different osmolytes, which demonstrates the ability of these regions to fold absent of a binding partner. This research presents new opportunities for investigating IDP structures.