Schleicher, Kai D.. Reduction of dimensionality in Karyopherinβ1 mediated transport on FG domains. 2014, PhD Thesis, University of Basel, Faculty of Science.
Available under License CC BY-NC-ND (Attribution-NonCommercial-NoDerivatives).
Official URL: http://edoc.unibas.ch/diss/DissB_11654
NPCs are biological nanomachines that regulate the selective exchange of macromolecular cargoes between the cytoplasm and nucleus in living cells. Small molecules diffuse freely through the NPC, whereas macromolecules >~5 nm in size are withheld. Access is limited to cargo-carrying transport receptors (karyopherins or Kaps, e.g. Kapß1), which interact with several intrinsically disordered Phe-Gly (FG)-repeat rich domains (i.e. FG domains) that pave the central pore. As each Kapß1 molecule contains ~10 hydrophobic pockets that bind FG repeats, Kap-FG domain binding involves highly multivalent interactions, which are generally known to impart a strong avidity that enhances stability and specificity. Consequently, in vitro studies have revealed very stable Kap-FG domain complexes. However, this is paradoxical in the context of the NPC, because the high Kapß1-FG domain binding affinities in the submicromolar range predict slow dissociation rates that contradict the short Kap-NPC dwell times measured in vivo (~5 ms). As this implies, Kap-FG domain binding ought to be sufficiently strong to ensure selectivity, but also weak enough to promote fast translocation through the NPC. However, an explanation as to how Kap-FG domain interaction balances the tradeoff between mobility and specificity during nucleocytoplasmic transport (NCT) is still lacking.
In the work presented here, this discrepancy is addressed in vitro using optical trapping-based photonic force microscopy (PFM). By measuring the thermal fluctuations of Kap-functionalized colloidal probes in contact with a surface grafted FG domain layer, it was found that Kap-FG interactions per se attenuate diffusive motion due to strong specific binding. This can be controlled by varying the amount of free Kaps in solution, which leads to differential behavior ranging from highly constrained to near-passive diffusion that is attributed to diminishing multivalent interactions between the Kap-probe and the FG domain layer. Measurements using surface plasmon resonance are consistent with this interpretation and show that a reduction of free FG-binding sites follows from a concentration-dependent increase in the occupancy of soluble Kapß1 molecules within the FG domain layer.
With the optical trap switched off, the probes exhibited two-dimensional diffusion at physiological Kap concentrations. The dissertation explains how multivalent interactions balance binding affinity and Kap-facilitated mobility on FG domains, leading to “reduction of dimensionality” in selective transport processes. This has implications for NCT, where a ROD-based scenario was proposed in which Kaps can diffuse in two dimensions along a layer of FG domains lining the central pore. Although this has not been validated in vivo, the physical display of Kap-facilitated two-dimensional diffusion on FG domains indicates that ROD can play a functional role in expediting selective transport through biological NPCs.
The importance and relevance of the work lie both in the understanding of multivalent interactions and multivalency-regulated transport processes in biological systems, as well as in breaking ground for the development of controlled reduced dimensional diffusion and controlled motion in artificial systems. On a more technical note, this work demonstrates the use of PFM in accessing particle diffusivity in the presence of biochemical interactions at biointerfaces.
|Advisors:||Lim, Roderick and Jeney, Sylvia|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Nanobiology Argovia (Lim)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||1 Online-Ressource (145 Seiten)|
|Last Modified:||14 Sep 2016 12:29|
|Deposited On:||25 Aug 2016 09:58|
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