Permeability-Based Measurement of Nanobody Binding to Nuclear Pore Complexes by Transient Scanning Electrochemical Microscopy

Abstract

Nucleocytoplasmic macromolecular transport through the nuclear pore complex (NPC) is mediated selectively and efficiently by protein–protein interactions, thereby regulating gene expression and the cell cycle. The NPC transport barrier is formed by biomolecular condensates of nucleoporins (nups) rich in hydrophobic phenylalanine–glycine (FG) repeats. Herein, we develop the transient mode of scanning electrochemical microscopy (SECM) to measure NPC interactions with a nanobody protein targeting the non-FG site of the crucial barrier-forming FG-rich nup, Nup98. The small nanobody, which was recombinantly expressed and purified, diffuses into the NPC and partially blocks the transport of an FG-binding polycationic peptide, protamine. The lowered permeability is measured more sensitively by transient SECM than by the traditional steady-state counterpart under our experimental conditions. A dissociation equilibrium constant between the nanobody and Nup98 in the authentic NPC is determined from the dependence of polypeptide permeability on nanobody concentration. The resulting dissociation constant of 7.7 nM is significantly higher than that of 1.7 nM measured for isolated Nup98 with the nanobody attached to the optical sensor surface in biolayer interferometry. The higher dissociation constant of nanobody–Nup98 interactions in the NPC is consistent with the enthalpically and entropically unfavorable partitioning of the nanobody into the condensate of hydrophobic and disordered nups. The powerful SECM method will be applicable to investigate a variety of NPC-binding proteins with the authentic NPC, thereby advancing our understanding of protein–NPC interactions and functions.