Abstract
Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule fluorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes.
Highlights
Disordered proteins (IDPs) usually fold during binding to target proteins
We investigated the molecular mechanism of such fast binding: association of the transactivation domain (TAD) of the tumor suppressor protein p53 and the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP)[15] using single-molecule FRET spectroscopy (Fig. 1a)
As a first step toward this direction, we employed photon-byphoton analysis for single-molecule FRET with high time resolution to systematically measure the lifetime of transient complex (TC) of TAD and NCBD as a function of ionic strength and describe the binding mechanism
Summary
Disordered proteins (IDPs) usually fold during binding to target proteins. In order to obtain accurate parameters, we used a maximum likelihood method that extracts the FRET efficiencies of the bound and unbound states and apparent association and dissociation rates[38] (see Supplementary Fig. 3 and Methods for the kinetic models and the details of analysis methods). The FRET efficiency of the unbound state (EU) gradually increases with the increasing NaCl concentration while that of the bound state (EB) remains unchanged, indicating that disordered TAD becomes more compact because of reduced electrostatic repulsion at higher ionic strength[39]. The association rate coefficient (kA) of a bi-molecular reaction (the reaction is pseudo-first order because [TAD] « [NCBD]), which is obtained by dividing kA,app by the NCBD concentration, decreases as the NaCl concentration is increased (Fig. 2c)
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