Complexes of charged disordered polyelectrolyte-like proteins: From thermodynamics to stuctural plasticity.

Abstract

The highly charged IDPs ProTα and H1 (−44 and +53, respectively) form a high-affinity, yet disordered complex and thus define a limiting case in the spectrum of disorder in IDP complexes [1-3]. Temperature-dependent single-molecule FRET (smFRET) experiments and isothermal titration calorimetry (ITC) reveal ProTα-H1 complexation to be enthalpically unfavorable. Using smFRET, we also identify stoichiometrically defined ternary complexes between ProTα and H1 at equilibrium and quantitate their salt-dependent stabilities, allowing us to accurately analyze ensemble experiments such as ITC performed with micromolar protein concentrations, where ternary complex formation is pronounced. Salt-dependent measurements show that counterion release entropy is a dominant driving force of complexation. An analytical polymer theory for polyelectrolyte complexation that explicitly accounts for counterions explains the thermodynamic results and suggests that the positive binding enthalpy is dominated by the relative exothermicities of counterion condensation and interchain ion-pair formation. Despite its high affinity, the disordered complex shows remarkable plasticity and expands continuously with increasing salt concentration, as seen from inter- and intramolecular FRET using various ProTα and H1 labeling variants. However, the salt-dependent expansion is non-uniform, which can be explained by differences in charge densities in different ProTα and H1 segments. Our investigations provide a comprehensive framework for the unconventional behavior of polyelectrolyte-like biomolecular complexes.