Resolving conformational changes in FG nucleoporins due to multivalent Karyopherin binding

The nuclear pore complex (NPC) is a huge macromolecular assembly that selectively regulates the exchange of cargo between the nuclear and the cytoplasmic compartment in a cell. The transport of cargo is mediated by nuclear transport receptors (NTR’s), such as Karyopherin, via interacting with NPC proteins (Nucleoporins or Nups) containing highly disordered phenylalanine-glycine (FG) domains. This thesis is an investigation into the structure – function relationship of FG domains, which are located within the nuclear pore complex (NPC) and thus are fundamental to the NPC gating mechanism. Surface plasmon resonance (SPR) is used to measure Karyopherin – FG domain binding affinity and kinetics in vitro near physiological NTR concentrations. To contribute structural information in situ a novel SPR based method is applied that uses non-interacting molecules as innate structural probes. A major part of the thesis is thus dedicated to the proof of concept of using non-interacting molecules to determine the height of surface tethered molecular brushes. The conformation of close-packed surface tethered FG domains reveals molecular brush like extension and is directly impacted by Karyopherin binding. The findings predict that unlike FG centric models NTR’s form a key component of the NPC and NTR-occupancy controls selectivity and speed of nucleo-cytoplasmic transport. This has the important implication that NTR – FG domain binding avidity due to multivalent interactions does not hinder fast transport per se.