Helix formation during the coupled binding and folding of intrinsically disordered proteins monitored by synchrotron-radiation circular dichroism spectroscopy

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signalling processes. These proteins often gain structure upon binding to their target proteins in multi-step reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain (NCBD) and the disordered interaction domain from activator of thyroid hormone and retinoid receptors (ACTR). We demonstrate that helix formation in a rapid binding reaction can be followed by stopped flow synchrotron-radiation circular dichroism spectroscopy, and describe the design of such a beamline. Fluorescence-monitored binding experiments of ACTR and NCBD display several kinetic phases including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time resolved circular dichroism experiments show that almost all helicity is formed upon initial association of the proteins, or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron radiation circular dichroism for mechanistic studies of protein-protein interactions.