Nanoscale Hydrophobic Interactions of Neurotoxic Dipeptide Repeats with the Nuclear Pore Complex as Revealed by Transient Scanning Electrochemical Microscopy

The goal of this thesis is to understand the role of the nuclear pore complex (NPC) as the sole gateway for nucleocytoplasmic transport of eukaryotic cells. Specifically, I will discuss how neurotoxic dipeptide repeats (DPRs) block the proteinous nanopore to potentially cause neurological diseases, e.g., amyotrophic lateral sclerosis and frontotemporal dementia. I will measure and quantitatively analyze the thermodynamic and kinetic interactions between NPC transport barriers and DPRs to understand how the NPC engages in selective and efficient molecular transport, which is biologically and biomedically important. To achieve these goals, transient scanning electrochemical microscopy (SECM) is developed, using a micrometer-sized electrode with an interface between two immiscible electrolyte solutions (ITIES) to probe DPR transfer across the liquid/liquid interface to determine the strength and kinetics of NPC–DPR interactions and concentration of interaction sites in the NPC. Chapter 1 is an overview of this thesis. Chapter 2 reviews recent developments and applications of nanoscale ITIES electrochemistry, including previous studies on monovalent ion diffusion through porous membranes and the NPC to establish the structure–permeability relationship, used in later chapters. In Chapter 3, the theory behind the transient method is modeled by the finite element method to determine the three parameters separately, instead of determining the overall permeability as the three combined using steady-state measurements. In Chapters 4 and 5, interactions of authentic NPCs with arginine-containing DPRs are quantified for the first time to reveal both cation-π and hydrophobic interactions. DPRs associate with the NPC as strongly as nuclear transport receptors but dissociate much more slowly to have longer dwell times, proposed as a potential cause of neurotoxicity. The length of DPRs is also varied to be used as molecular probes to engage in hydrophobic interactions with the transport barriers, revealing that larger DPRs associate more strongly with and remain longer to clog NPCs, supporting the hypothesis of NPC transport utilizing hydrophobic interactions. Similar free energy changes determined for transfer into the NPC and nitrobenzene suggest their similar hydrophobicity. This demonstrates the potential for liquid/liquid interface to model other hydrophobic proteinous environments beyond bilayer lipid membranes, as exemplified by NPC transport barriers.