Figure 1.
The yeast nuclear pore complex, some of its FG nucleoporins, and an ensemble of protein structures generated by MD simulations of a Nup116 FG domain.
(A) A simplified diagram of the yeast nuclear pore complex (green), its cytoplasmic fibrils (yellow), its nuclear basket (red) and some of the GLFG-rich domains of nups that line the transport conduit (blue). The GLFG-rich domains are depicted as a doughnut-shaped array of laterally-cohesive, native pre-molten globules. Other FG domains are excluded for simplicity. The nuclear envelope is in gray. (B) GLFG-rich nucleoporins in the yeast NPC transport conduit. The vertical tick marks in the nups mark the location of each FG motif: GLFG motifs are in yellow, FxFG motifs in red, and other variants in different colors. The fragment of Nup116 (AA 348–458) indicated was selected as a representative FG domain for this study. The F>A mutant version lacks the phenylalanine residue in FG motifs, which were replaced by alanine. (C) The AA sequences for the wild-type and mutant Nup116 FG domains used for MD simulations. The phenylalanine (or alanine) in FG motifs is indicated in red. The AA sequences in gray are not part of Nup116; they are affinity-tags used in the purification of the FG domain. The numbers in the black bold font indicate the AA position in the Nup116 sequence. The numbers in gray indicate the AA position in the FG domain fragment analyzed. The protein structures shown are a representative subset of the twenty MD simulation structures generated for each FG domain at 350 K. The Rg values of the representative structures are within the average range for each FG domain (see Figure 2A).
Figure 2.
Molecular dimensions of the simulated FG domain structures.
(A) Average radii of gyration of Nup116 FG domains simulated at different simulation temperatures. Box-plot of average radii of gyration (Rg) in units of Angstroms calculated from twenty replicate 1 ns simulations at 300, 325, and 350 K for the wild-type and F>A mutant FG domains. (B) Histogram of radii of gyration (calculated using only the atoms in the peptide backbone) for the 10,000 FG domains structures sampled from the 350 K simulations. (C) Histogram of end-to-end distances (calculated from the terminal C and N atoms) for 10,000 FG domain structures obtained by sampling every 1 ps of the final 500 ps of each of the twenty replicate MD simulations at 350 K.
Figure 3.
Interresidue distances in the simulated FG domains.
(A) Plots of the probability distribution of inter-atomic distances in the wild-type and mutant FG domains. The distance distribution between the backbone β-carbons of phenylalanine (F) or alanine (A) residues in positions 84 and 93 (see Figure 1C) is shown as a representative example. The solid line in each plot corresponds to a Gaussian fit to the probability distribution. (B) Pearson squared correlation plots of the atomic distance between F–F or A–A pairs in the wild-type and mutant Nup116 FG domains. The numbers in the axes correspond to the various F–F or A–A pairs that result from all possible combinations (listed in Table S1). The correlation map shows how each of the pairs is related to the others. The insert depicts the contour level of the Pearson coefficient. (C) Schematic representations of Phe-to-Phe distances in the wild-type FG domain and Ala-to-Ala distances in the F>A mutant domain. The calculated average distance between the Phe or Ala residues in the pair-wise combinations is thickness and color coded. Thick red lines represent distances between 10 and 15 Å; medium blue lines represent distances between 15 and 20 Å; and thin green lines represent distances greater than 20 Å.
Figure 4.
Analysis of Nup116 FG domains by NMR.
Left panels: Aromatic and amide region of the water-gate residual water suppressed one-dimensional NMR spectra for the purified FG domains. Tall peaks in the spectrum between 7.1 and 7.3 ppm arise from the Phe residues in the wild-type domain, which are absent in the F>A mutant. The sensitivity of the mutant FG domain spectrum is lower due to a lower protein concentration than the wild-type FG domain. Right panels: Plot of the self-diffusion coefficient measurements performed using BPP-SED for the wild-type and mutant FG domains. Circles depict the experimental points and squares, and lines correspond to the fit to the diffusion data. The error bars are smaller than the size of the symbols.
Table 1.
Hydrodynamic dimensions of the purified Nup116 FG domains.