Evidence that small proteins translocate through silicon nitride pores in a folded conformation

Abstract

The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiN(x)) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5-1.0 nm.

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore

“…A likely explanation for the observed voltage dependences, as previously reported in the literature for other moleules, [42][43][44][45][46] is that at potentials lower than the dwell time maximum (−40 mV for Type I ClyA-AS and −30 mV for Type II ClyA-CS) the majority of DHFR ST proteins enter and exit the nanopore from the cis side, while at potentials above the dwell time maximum the majority of proteins translocate through the nanopore (Figure 2c,d). (Figure 3b,c, step 3, Figure S4b, Figure S5).…”

Nanoscale Investigation of Generation 1 PAMAM Dendrimers Interaction with a Protein Nanopore

“…4, panels c and d). The indirect indication relating analyte translocation by its voltage-dependence dissociation from the nanopore, as used above, was also described and discussed for the case of other analytes 50–55 . Note that for the larger PAMAM-G2 dendrimer, at both pH’s, an increase in the applied potential led to an increase of the dendrimer’s dissociation time (τ off ) (Fig.…”

Nanopore analysis

“…Recent experiments have shown that folded proteins give smaller blockade currents than the corresponding random coil but it is not clear that different conformations would yield different signatures since the occluded volumes would be very similar. 70 There are also problems with the reproducibility of solid state pores since it is very difficult to control the diameter during fabrication and the chemistry of the internal surfaces can be quite variable. One elegant solution is to coat the pore with a lipid.…”