Nanoscale Hydrophobicity of Transport Barriers in the Nuclear Pore Complex as Compared with the Liquid/Liquid Interface by Scanning Electrochemical Microscopy.

The nuclear pore complex (NPC) is the proteinous nanopore that solely regulates molecular transport between the nucleus and cytoplasm of a eukaryotic cell. Hypothetically, the NPC utilizes the hydrophobic barriers based on the repeats of phenylalanine-glycine (FG) units to selectively and efficiently transport macromolecules. Herein, we quantitatively assess the hydrophobicity of transport barriers confined in the nanopore by applying scanning electrochemical microscopy (SECM). The hypothesis deduced from studies of isolated FG-rich nucleoporins is supported quantitatively by investigating the authentic NPC for the first time. Specifically, we employ the n repeats of neurotoxic glycine-arginine dipeptide, GR(n), as the molecular probes that engage in hydrophobic interactions with transport barriers in the NPC. We apply ion-transfer voltammetry at a micropipet-supported interface between aqueous and organic electrolyte solutions to confirm that larger GR(n) among n = 5-25 is more hydrophobic, as expected theoretically. The micropipet also serves as the tip of transient SECM to demonstrate that the NPC interacts more strongly with larger GR(n), which supports the hydrophobicity of transport barriers. Kinetically, larger GR(n) stays in the NPC for longer to clog the nanopore, thereby expressing neurotoxicity. Significantly, this work implies that the efficient and safe nuclear import of genetic therapeutics requires an optimum balance between strong association with and fast dissociation from the NPC. Interestingly, this work represents the unexplored utility of liquid/liquid interfaces as models of hydrophobic protein condensates based on liquid-liquid phase separation as exemplified by nanoscale transport barriers in the NPC.