Conformational changes at supramolecular interfaces are fundamentally coupled to binding activity, yet it remains a challenge to probe this relationship directly. Within the nuclear pore complex, this underlies how transport receptors known as karyopherins proceed through a tethered layer of intrinsically disordered nucleoporin domains containing Phe-Gly (FG)-rich repeats (FG domains) that otherwise hinder passive transport. Here, we use nonspecific proteins (i.e., BSA) as innate molecular probes to explore FG domain conformational changes by surface plasmon resonance. This mathematically diminishes the surface plasmon resonance refractive index constraint, thereby providing the means to acquire and correlate height changes in a surface-tethered FG domain layer to Kap binding affinities in situ with respect to their relative spatial arrangements. Stepwise measurements show that FG domain collapse is caused by karyopherin β1 (Kapβ1) binding at low concentrations, but this gradually transitions into a reextension at higher Kapβ1 concentrations. This ability to self-heal is intimately coupled to Kapβ1-FG binding avidity that promotes the maximal incorporation of Kapβ1 into the FG domain layer. Further increasing Kapβ1 to physiological concentrations leads to a "pileup" of Kapβ1 molecules that bind weakly to unoccupied FG repeats at the top of the layer. Therefore, binding avidity does not hinder fast transport per se. Revealing the biophysical basis underlying the form-function relationship of Kapβ1-FG domain behavior results in a convergent picture in which transport and mechanistic aspects of nuclear pore complex functionality are reconciled.