Fig. 4: Controlled immobilization of TOM-CC triggers channel closures.
a Schematic representation of individual TOM-CC channels in DIB membranes supported by Ni-NTA-modified agarose. TOM-CC molecules can be permanently linked to the underlying hydrogel via His-tagged Tom22. Tethered and non-tethered TOM-CC molecules in closed (SI and SL) and open (SH) states are indicated, respectively. b Fluorescent amplitude trace (top) of a TOM-CC channel permanently tethered to Ni-NTA-modified agarose. The trajectory segments (bottom) correspond to the time periods of the amplitude traces marked in gray (Supplementary Movie S7: bottom). Non-diffusive, permanently immobilized TOM-CC is only found in SI or SL, indicating that tight binding of the His-tagged Tom22 domain (Fig. 1b) to Ni-NTA-modified agarose triggers closure of the β-barrel TOM-CC pores. c Fluorescence amplitude trace (top) of a TOM-CC channel transiently and non-specifically entangled by Ni-NTA-modified agarose. The trajectory segments (bottom) correspond to the time periods of the amplitude traces marked in gray. The movement of TOM-CC is interrupted twice at the same spatial x,y membrane position from t1 = 18.56 s to t2 = 19.19 s and from t3 = 31.14 s to t4 = 34.55 s (yellow stars). Consistent with the data shown in Fig. 3, moving TOM-CC molecules in diffusive mode are found in the fully open SH state; transient tethering causes the TOM-CC β-barrels to close. Data were acquired as described in Fig. 1c at a frame rate of 47.5 s−1. A total of nTOM = 123 amplitude traces and trajectories were analyzed. a.u. arbitrary unit.
