Molecular Origins of Free Energies Associated with Complex Interactions of Biological Polyelectrolyte-Like Disordered Proteins

Abstract

Diverse molecular forces drive the interactions of intrinsically disordered proteins (IDPs) and result in complexes that cover a broad spectrum of structural plasticity. Prothymosin-α (ProTa) and linker Histone (H1) are two IDPs that bear large negative and positive net charges, respectively, and form a picomolar-affinity yet disordered complex [1] - the limiting case in the spectrum of structural plasticity in IDP complexes. Here we show that their interaction exhibits remarkable thermodynamic behavior. Single-molecule FRET-based affinity measurements as a function of salt concentrations and temperature show that the predominant driving force of binding is the entropy increase due to counterion release upon complexation. As a result, the affinity increases with temperature and is extremely sensitive to ionic strength. Single-molecule FRET also enables the identification of stoichiometrically defined ternary complexes of ProTa and H1 at equilibrium and how their stability depends on solution conditions. These ternary complexes have pronounced effects on the interaction kinetics of ProTa-H1 [2], and they complicate affinity measurements in ensemble experiments, because at the much higher protein concentrations required compared to single-molecule conditions, the observed apparent affinities contain contributions from both binary and ternary complexes. [1] Borgia et al., Extreme disorder in an ultrahigh-affinity protein complex (2018), Nature 555, 61-66. [2] Sottini et al., Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes (2020), Nature Communications (in press).