Mapping Multivalency and Differential Affinities within Large Intrinsically Disordered Protein Complexes with Segmental Motion Analysis

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time‐resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region‐specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes—a result with implications for the diversity of transport routes in nucleocytoplasmic transport. Flexible backbones: A combined fluorescence correlation and time‐resolved polarization spectroscopy approach was used to study differential binding within large and disordered protein complexes with multivalent interactions. The differential segmental backbone mobility of a disordered nucleoporin in complex with transport receptors was detected. Local binding preferences were identified within the disordered protein which range over several orders of magnitude.