Figure 1. Isotherms of NTR binding (top row, log-log presentation) and FG domain film thickness evolution (bottom row, lin-log presentation) for NTF2 and Impβ binding to different FG domains (see labels at top) at selected FG domain grafting densities (visualized by distinct symbols and colors).
Error bars are shown for all data points in the binding isotherms, and for three selected data points (indicating the trends) per curve in the thickness isotherms. The data for Impβ binding to the 10.0 pmol/cm2 Nsp1 film were reproduced from Eisele et al. (2010); this data was acquired with Nsp1 carrying a His tag at the opposite end (N-terminus) compared to the other Nsp1 data in this study, in a separate SE measurement and no simultaneously recorded thickness data are available. Full experimental details are available in ‘Materials and methods’ and Figure 1—figure supplements 1–5; tabulated results are available in Figure 1—source data 1.
DOI: http://dx.doi.org/10.7554/eLife.14119.005
Figure 1—source data 1. Tables of data shown in Figure 1.
elife-14119-fig1-data1.xlsx (17.7KB, xlsx)
DOI: 10.7554/eLife.14119.006
Figure 1—figure supplement 1. Quality of purified recombinant proteins used in this study.
FG domains with His tag were dissolved in formamide and diluted 1:3 in SDS sample buffer. 1.5 to 2.5 μg of each protein construct were resolved by SDS-PAGE and stained with Coomassie G250. The band corresponding to reg-SSSG runs much higher than the molecular weight expected; this is usually the case for very hydrophilic proteins (Shirai et al., 2008). All preparations contain more than 95% full length protein.
Figure 1—figure supplement 2. FG domains are anchored specifically and stably through their terminal His tag.
(A) Binding and elution of reg-FSFG was monitored by SE, on a silica surface previously functionalized with a supported lipid bilayer (SLB; 7% tris-NTA). Arrows on top of the graph indicate the start and duration of incubation with different sample solutions; during remaining times, the surface was exposed to working buffer. Most of the reg-FSFG remains stably bound upon rinsing in working buffer. reg-FSFG was fully eluted after the imidazole treatment (grey shaded area, not monitored), demonstrating that binding is specific through the His tag. (B-D) Site-specific and stable anchoring of Nsp1 to His tag capturing QCM-D sensors, as well as Nsp1 and Nup98-glyco to SLBs (10% bis-NTA) on silica is demonstrated by QCM-D; the data are reproduced from Figure 1 in Eisele et al. (2012) and Figure S2 in Eisele et al. (2013), respectively.
Figure 1—figure supplement 3. Schematic illustration of the experimental approach and representative data.
(A) Schematic illustration of the experimental approach. (B–C) Representative data for combined SE/QCM-D measurements. Areal protein densities (top) are obtained through optical modeling of SE data (see Materials and methods). QCM-D responses (bottom) are recorded in parallel, and normalized frequency shifts Δfi/i and dissipation shifts ΔDi for a selected overtone (i = 3) displayed here. Film thickness is obtained through viscoelastic modeling of QCM-D data (see Materials and methods). Δf = ΔD = 0 corresponds to the functionalized surface before FG domain grafting. (B) Monitoring of FG domain film formation. Nsp1 was exposed to a silica substrate previously functionalized with an SLB (7% tris-NTA). The final grafting density is 4.9 pmol/cm2. Minor perturbations in Δf and ΔD between 33 and 43 min are due to transient variations in the solution temperature in contact with the QCM-D sensor during the rinsing with buffer, and do not represent changes of the Nsp1 film. (C) Time-resolved data for the titration of NTF2 into this Nsp1 film. The NTF2 solution concentration was increased in 12 steps (0.0025, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 5, 7.5 and 10 μM), and then decreased in 16 steps ((2/3)j × 10 μM, with j = 1, …, 16), followed by continuous rinsing with buffer solution to remove all NTF2 from the bulk solution. The rapid binding and unbinding of NTF2 observed here is representative for all titration measurements performed, and binding equilibriums could thus be readily attained. Moreover, binding of NTF2 was largely reversible, with less than 7% of the maximal binding remaining following rinsing in buffer for any given measurement. For Impβ, more than 75% were readily eluted from 5 pmol/cm2 Nsp1 films, and we had previously shown this NTR to elute close to completely from 10 pmol/cm2 Nsp1 films (Eisele et al., 2012; Eisele et al., 2010).
Figure 1—figure supplement 4. Controls for the binding of NTRs to His tag capturing surfaces monitored by QCM-D.
(A) NTF2 on His tag capturing QCM-D sensor; (B) NTF2 on SLB (7% tris-NTA); (C) Impβ on SLB (4% tris-NTA). The vertical scales were chosen such that the full range would approximately cover the magnitude expected for a full monolayer of NTRs.
Figure 1—figure supplement 5. NTF2 binds all FG domains predominantly through its primary binding site.
(A) Ratio of equilibrium bound amounts of NTF2 W7A mutant and wild type NTF2 as a function of FG domain type. NTF2 and NTF2 W7A mutant were sequentially exposed to FG domain films, and binding was quantified by SE. Mean and standard errors of two to four independent measurements per FG domain type with FG domain surface densities ranging between 5 and 13 pmol/cm2 are presented. The tryptophan at position 7 is known to be important for the binding of NTF2 through a structurally defined (Bayliss et al., 2002) binding site to FG motifs (Bayliss et al., 1999). For all tested FG domain types, binding of the W7A mutant was reduced by more than 80% compared to native NTF2. (B) Native NTF2 did not bind to reg-SSSG (here at 8.3 pmol/cm2), confirming that binding to reg-FSFG requires the FSFG motif. These data confirm that the NTF2 in our experiments binds specifically to the immobilized FG domains through its primary FG motif binding site.