Analysis of multivalent IDP interactions: Stoichiometry, affinity, and local concentration effect measurements

Abstract

Nuclear magnetic resonance (NMR) titration and isothermal titration calorimetry can be combined to provide an assessment of how multivalent intrinsically disordered protein (IDP) interactions can involve enthalpy–entropy balance. Here, we describe the underlying technical details and additional methods, such as dynamic light scattering analysis, needed to assess these reactions. We apply this to a central interaction involving the disordered regions of phe–gly nucleoporins (FG-Nups) that contain multiple phenylalanine–glycine repeats which are of particular interest, as their interactions with nuclear transport factors (NTRs) underlie the paradoxically rapid yet also highly selective transport of macromolecules mediated by the nuclear pore complex (NPC). These analyses revealed that a combination of low per-FG motif affinity and the enthalpy–entropy balance prevents high-avidity interaction between FG-Nups and NTRs while the large number of FG motifs promotes frequent FG–NTR contacts, resulting in enhanced selectivity.