Figure 3.

Pre-incubation of the FG/FxFG hydrogel with scImpβ tightens the barrier against passive influx. Panels A–C show concentration profiles of mobile species during their entry into a saturated FG/FxFG_2–601^Nsp1 hydrogel. The three time points (30 s, 10 min and 30 min) are colour-coded. For best comparison, the free concentration of the mobile species in buffer was scaled to 1. (A) Influx of MBP-mCherry alone. The comparison of the three time points indicates a slow, but still clearly detectable influx and a partition coefficient between gel and buffer of 0.18. (B) Simultaneous influx of 3 μM of MBP-mCherry and 1 μM of an IBB-MBP-mEGFP·scImpβ complex. The NTR·cargo complex entered the gel ≈100 times faster than the passive species. The presence of the NTR·cargo complex did not increase influx of the passive species as compared with panel A. (C) The influx experiment was performed as in panel B, the difference being that the FG hydrogel had been pre-incubated for 180 min with 10 μM of unlabelled, cargo-free scImpβ before the fluorescent mobile species was added. The pretreatment had only a minor effect on influx of the NTR·cargo complex (slight reduction in entry rate and intra-gel diffusion coefficient), indicating that this type of FG hydrogel is very robust against competition and can sustain a very high load of facilitated transport. However, the pretreatment had the striking effect of tightening the barrier against passive influx, and thus improving the performance of the barrier greatly. The labelled scImpβ·cargo complex now entered the gel at least 5000 times faster than the inert reference molecule MBP-mCherry. (D) Analytical gel filtration revealed a Stokes radius (R_S) of 3.6 nm for the above used inert reference molecule (MBP-mCherry) and 5.1 nm for the IBB-MBP-mEGFP·scImpβ complex.