Spatial structure of disordered proteins dictates conductance and selectivity in nuclear pore complex mimics
- Adithya N Ananth
- Steffen Frey
- Arvind Dwarkasing
- Roderick Versloot
- Erik van der Giessen
- Dirk Görlich
- Patrick Onck
- Cees Dekker
- Delft University of Technology, Netherlands
- University of Groningen, Netherlands
- Max Planck Institute for Biophysical Chemistry, Germany
Figures
Figure 1 with 7 supplements
Coating a nanopore with FG-Nups reduces the pore conductivity.
(A) Schematic of the biomimetic NPC where yeast FG-Nup Nsp1 is coated onto a solid-state nanopore of diameter 50 nm and thickness of 20 nm. Kap95, a yeast importer, can pass through the barrier, …
Figure 1—figure supplement 1
Self-assembled monolayer surface chemistry for covalently attaching Nsp1 and Nsp1-S to Silicon Nitride membrane.
The APTES layer was used as a primary monolayer through silanization. Next, the NHS-ester with a maleimide reactive group (Sulfo-SMCC) was coupled to the APTES monolayer. The exposed maleimide …
Figure 1—figure supplement 2
Current power spectral density of a bare pore and nanopores coated with Nsp1 and Nsp1-S, versus frequency.
From the spectrum of the Nsp1 pore it is evident that the 1/f noise increases drastically compared to the bare pore (Smeets et al., 2008). This is a clear indication of the fluctuations of Nsp1. A …
Figure 1—figure supplement 3
Transmission electron microscopy (TEM) images of bare and Nsp1-coated pores.
(A, B) 30 nm pores and (C,D) 50 nm pore before and after Nsp1 coating respectively. While these images of dried pores do not necessarily represent the intrinsic mass distribution, they serve to …
Figure 1—figure supplement 4
Histogram of the conductance of individual Nsp1 molecules translocating through a bare pore, and the associated scatter plot of conductance versus translocation time.
https://doi.org/10.7554/eLife.31510.006
Figure 1—figure supplement 5
Histogram of the conductance of individual Nsp1-S molecules translocating through a bare pore, and the associated scatter plot of conductance versus translocation time.
https://doi.org/10.7554/eLife.31510.007
Figure 1—figure supplement 6
Example QCM-D traces for the surface functionalization of Nsp1 (A) and Nsp1-S (B) on silicon nitride coated with APTES and Sulfo-SMCC.
https://doi.org/10.7554/eLife.31510.008
Figure 1—figure supplement 7
Average protein densities of a Nsp1-S coated solid-state nanopore of diameter 45 nm with grafting distance 5.7 nm (green) and 5.9 nm (red).
https://doi.org/10.7554/eLife.31510.009
Figure 2 with 2 supplements
Coarse-grained molecular dynamics results of Nup density distributions in Nsp1 and Nsp1-S pores of varying diameter.
(A) Coarse-grained one-bead-per-amino-acid representation of Nsp1; the different colors of the beads represent the 20 different amino acids. The collapsed-coil N-terminal ‘head’ region is visible at …
Figure 2—figure supplement 1
Top: The configuration of Nsp1 consists of a ‘collapsed coil’ head (in blue) and a ‘extended coil’ stalk region (multi-colored; each color represents a different amino acid).
Bottom: Two-dimensional r-z density distribution of the mass density of the head-region (1-172) of Nsp1, for nanopores with diameters 22 nm, 45 nm and 60 nm (first row). The second row shows the …
Figure 2—figure supplement 2
Simulation results for the density distribution in Nup98-coated biomimetic pores.
Top panel: Two-dimensional r-z density distribution of Nup98-coated pores with diameters 22 nm, 45 nm, and 65 nm. Bottom panel: the corresponding radial density distributions. All data are taken …
Figure 3 with 4 supplements
Radial density distribution and conductance data for Nsp1 and Nsp1-S biomimetic pores.
(A) Radial protein density distribution for biomimetic nuclear pores with pore diameters of 22 nm, 45 nm, and 60 nm, for pores coated with Nsp1 (blue) and Nsp1-S (green). All data are taken within …
Figure 3—figure supplement 1
Conductance as a function of pore diameter below 40 nm.
The conductance change is plotted versus the diameter of Nsp1 coated pores. The measured conductance of the NPC falls in the range of 0.3–2 nS for monovalent salt (Bustamante et al., 1995; Tonini et …
Figure 3—figure supplement 2
The conductivity in the pore region () and access region () for the simulated nanopores lined with Nsp1 (blue circles; panel A) and Nsp1-S (green circles; panel B) plotted as a function of pore diameter.
For the density distribution is integrated over the pore region |z| < 10 nm according to Equation 3 in the main text. For the conductivity in the access region the density distribution in the …
Figure 3—figure supplement 3
The conductivity and conductance for the simulated nanopores lined with Nup98 as a function of pore diameter.
(A) Based on the radial density distributions of the Nup98 pore (Figure 2—figure supplement 2, bottom row), we calculated the pore conductivities and (black circles) for each diameter using …
Figure 3—figure supplement 4
Computed conductance vs the experimentally measured conductance, for (A) Nsp1 (blue circles) and Nsp1-S (green circles), and (B) Nup98 (black circles).
The solid lines have a slope of 1, representing a perfect match between experiment and model.
Figure 4
Transport and selectivity of the biomimetic NPCs.
(A) Typical translocation event of Kap95 through a bare pore. Each spike signals a single kap95 that translocate the pore. (B) Examples of translocation events through the Nsp1-coated pores. Note …
Figure 5 with 3 supplements
Selectivity for Nsp1, but not for Nps1-S biomimetic NPCs.
(A) Event frequencies for Kap95 (blue) and tCherry (red) through bare pores, Nsp1-coated pores , and Nsp1-S-coated pores. The data show an NPC-like selectivity for Nsp1-coated pores where the …
Figure 5—figure supplement 1
Stable baseline conductance and increase of the event rate with Kap95 concentration for an Nsp1-coated pore.
These experiments were carried out on Nsp1-coated pores with a varying Kap95 concentration in the buffer. (A) Baseline ionic conductance versus Kap95 concentration. Triangles and diamonds denote two …
Figure 5—figure supplement 2
Event frequency versus diameter for Kap95 and tCherry through Nsp1-S coated nanopores.
The event frequencies are averaged over different pores (n = 3). The error bars indicate standard deviations. A black cross is shown when no translocation events could be measured (e.g. because the …
Figure 5—figure supplement 3
Experimental event rate versus the computed energy barrier ∆E, for tCherry and Kap95 in Nsp1 and Nsp1-S pores.
The data points are plotted as black circles and fitted using the Arrhenius relation (see Equation 4 in the main text). From the fitting we obtain = 16.4 Hz with an R2 value of 0.96.
Figure 6
Potential of mean force (PMF) curves associated with transport of tCherry and Kap95 through Nsp1 and Nsp1-S coated pores.
PMF values for tCherry and Kap95 particles at different positions along the central axis (r = 0). Kap95 in the Nsp1 and in Nsp1-S pore is represented in black and red, respectively, and tCherry in …
Videos
Video 1
A short trajectory of a coarse-grained one-bead-per-amino-acid molecular dynamics simulation of a biomimetic nanopore with a diameter of 45 nm coated with Nsp1, corresponding to Figure 2C (top row) of the main manuscript.
Only half the simulation box is shown for better visibility. The movie is prepared with the Visual Molecular Dynamics (VMD) software.
Video 2
A short trajectory of a coarse-grained one-bead-per-amino-acid molecular dynamics simulation of a biomimetic nanopore with a diameter of 45 nm coated with the mutant Nsp1-S, corresponding to Figure 2C (bottom row) of the main manuscript.
Only half the simulation box is shown for better visibility The movie is prepared with the Visual Molecular Dynamics (VMD) software.
Video 3
A short trajectory of a coarse-grained one-bead-per-amino-acid molecular dynamics simulation of an isolated Nsp1.
The cohesive head group at the N-terminus is located at the right. The movie is prepared with the Visual Molecular Dynamics (VMD) software.
Video 4
A short trajectory of a coarse-grained one-bead-per-amino-acid molecular dynamics simulation of an isolated mutant Nsp1-S.
The C-terminus is located at the top. The movie is prepared with the Visual Molecular Dynamics (VMD) software.
Additional files
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Supplementary file 1
Measured hydrodynamic diameters (nm) of proteins from dynamic light scattering (DLS) experiment.
- https://doi.org/10.7554/eLife.31510.028
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Supplementary file 2
Molecular characteristics of Nsp1 (see Video 3), Nsp1-S (see Video 4) and Nup98 and the computed Stokes radii (in isolation).
- https://doi.org/10.7554/eLife.31510.029
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Transparent reporting form
- https://doi.org/10.7554/eLife.31510.030
