Permeability-Based Measurement of Nanobody Binding to Nuclear Pore Complexes by Transient Scanning Electrochemical Microscopy.
- 1Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania15260, United States.
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.
Related
- Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes
- Editorial Note: Aquaporin expression contributes to human transurothelial permeability in vitro and is modulated by NaCl
- Single cell RNA-seq analysis identifies junctional adhesion molecule two positive endothelial cells aggravate osteoarthritis by mediating vascular permeability and macrophage adhesion
- Enterovirus A71 exploits peripheral circulating extracellular vesicles to facilitate blood-brain barrier permeability
- Condensin upregulation induces intestinal stem cell senescence, driving cell death and increased intestinal permeability
BibTeX
@article{parandhaman2026permeabilitybased,
title = {{Permeability-Based Measurement of Nanobody Binding to Nuclear Pore Complexes by Transient Scanning Electrochemical Microscopy.}},
author = {Moghitha Parandhaman and Siao-Han Huang and Kathryn G Dzurik and W Seth Childers and Shigeru Amemiya},
journal = {Analytical chemistry},
year = {2026},
month = oct,
doi = {10.1021/acs.analchem.6c04319},
url = {https://europepmc.org/article/MED/42899530},
note = {CC BY},
}
RIS
TY - JOUR
TI - Permeability-Based Measurement of Nanobody Binding to Nuclear Pore Complexes by Transient Scanning Electrochemical Microscopy.
AU - Moghitha Parandhaman
AU - Siao-Han Huang
AU - Kathryn G Dzurik
AU - W Seth Childers
AU - Shigeru Amemiya
PY - 2026
DA - 2026/10/01
JO - Analytical chemistry
DO - 10.1021/acs.analchem.6c04319
UR - https://europepmc.org/article/MED/42899530
AB - 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.
SN - 0003-2700
SN - 1520-6882
ER -