Single-Molecule FRET Probes a Transient Complex that Facilitates Binding of an Intrinsically-Disordered Protein

Abstract

Intrinsically-disordered proteins (IDPs) are unfolded at the native condition and fold only when attaching to their binding partners. Even though IDPs often undergo large conformational changes, which may slow the binding process, the binding kinetics are sometimes surprisingly fast, close to those of diffusion-controlled reactions. IDPs usually contain a large number of charged residues and theory predicts that the association can be facilitated by the formation of a transient complex (TC) stabilized by the electrostatic attraction between an IDP and its binding target. Although the modulation of the association rate by ionic strength of the solution has been observed for a number of IDPs, TC has not been directly probed during the course of binding. To experimentally investigate the role of TC in fast binding, we employed single-molecule Forster resonance energy transfer (FRET) spectroscopy that monitors the conformational evolution of an IDP from the unbound state to the bound complex. We studied binding of the transactivation domain (TAD) of the tumor suppressor protein p53 (negatively charged, −10) and the nuclear co-activator binding domain (NCBD) of CBP (positively charged, +6). The N- and C-termini of TAD were labeled with Alexa 647 and Alexa 488 site-specifically and molecules were immobilized on a glass surface using a biotin-neutravidin linkage to monitor binding to unlabeled NCBD in solution. Because of the fast binding kinetics, the bound state, TC, and unbound state could not be clearly resolved in binned trajectories. Instead of binning, we used the maximum likelihood method (Gopich and Szabo, JPCB(2009)) to analyze photon trajectories collected at high illumination intensity and to measure the binding kinetics (photon count rate of 50 - 100 ms−1) and the lifetime of TC (photon count rate of 500 - 1000 ms−1). We found that the association rate reaches ∼ 1 x 109 M−1s−1, which is close to the diffusion-limited rate, and the lifetime of TC is relatively long (∼100 microsecond) compared to the kinetics at low ionic strength (∼ 0.04 M). More importantly, both quantities decrease significantly with the increasing ionic strength, indicating that the destabilization of TC results in the slow association. This result demonstrates that the fast association of an IDP can be achieved by stabilizing TC through native/non-native electrostatic interactions, which would allow longer time for an IDP to interact with a target protein to fold and bind.