Paper BI+AS+BA+NL-TuM10
QCM-D as a Novel Technique to Investigate Nuclear Pore Transport
Tuesday, October 29, 2013, 11:00 am, Room 201 B
A quartz crystal microbalance (QCM-D) is a simple and highly sensitive mass and dissipation sensor which has been used to study interfacial adsorption reactions and conformational changes on a variety of supports in real time. In this paper we aim to apply this technique to gain a better understanding of nuclear transport. In particular, we are investigating the transport of proteins through the Nuclear Pore Complex (NPC), which is the sole mediator of exchange between the nucleus and the cytoplasm in all eukaryotic cells1. Recent publications have further improved our understanding of the architecture and evolutionary origins of this macromolecular gate,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. In an attempt to better understand the transport and the selective process under crowding conditions, we immobilized Nsp1 and truncated variations of it onto QCM-D sensors. The binding and unbinding of Kap95, other binding proteins, as well as control proteins (e.g. BSA), was studied in order to investigate specificity, kinetics rate constants, effect of competitive binding. Ultimately 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