(15d) Exploring the Specificity of Nuclear Pore Transport Using AFM and QCM-D
The Nuclear Pore Complex (NPC) is the sole mediator of exchange between the nucleus and the cytoplasm in all eukaryotic cells1. Its architecture is well understood and described in the literature,2,3 yet the molecular transport mechanism remains unclear. Transport across the NPC is fast, energy-dependent (to give directionality) and often receptor-mediated. While small molecules pass through the NPCs unchallenged, large macromolecules (>40 kDa) are excluded unless chaperoned across by transport factors collectively termed Karyopherins (Kaps). The translocation of the complexes of Kaps and their cargo proteins/RNAs occurs through the specific affinity and binding between Kaps and particular nuclear pore complex proteins (nucleoporins) called FG-Nups, which share a degenerate multiple-repeated “Phe-Gly” motif. Because FG-Nups are the major component of the selective gating mechanism, we first investigated the nanomechanical properties of cysteine-modified Nsp1 using the volume force mapping technique of atomic force microscopy (AFM). From single molecule AFM on a sparse Nsp1 surface, we estimated structural parameter as persistence length and contour length. In an attempt to better understand the transport and the selective process under crowding conditions, we then used quartz crystal microbalance with dissipation (QCM-D). Nsp1 and truncated variations of it were immobilized on QCM-D sensors. The binding and unbinding of Kap95, other binding proteins, as well as control proteins, was studied in order to investigate specificity, kinetics rate constants, effect of competitive binding. Inspired by Nature, we aim to gain sufficient understanding of the molecular scale engineering principles behind nuclear transport to allow us to design the next generation of synthetic selective nanosorters capable of purifying any protein that we desire.
1. Grünwald, Singer and Rout, Nature 2011, 475, 333.
2. Alber et al., Nature 2007, 450, 683
3. Alber et al., Nature 2007, 450, 695