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. 2017 Dec 5;139(51):18640–18646. doi: 10.1021/jacs.7b10106

Figure 3.

Figure 3

Orientation and dynamics of SBD2 measured by nanopore experiments. (A) Typical current blockade provoked by the capture of SBD2 (72 nM, cis) by the ClyA-AS nanopore. Left, apo-SBD2, middle current blockades after the addition of 0.40 μM of glutamine (cis); and right, blockades after adding 50 μM glutamine (cis). (B) Typical current blockades provoked by the capture of inactive SBD2(D417F) (70 nM) before and after the addition of 200 μM glutamine to the cis side. Red asterisks represent the restoration of the open pore current after SBD2(D417F) exited from the pore. (C) Kdapp value of SBD2 for glutamine obtained by fitting to a binding isotherm, using the relative closed population [LC/(LOA + LOB + LC)] at the indicated substrate concentrations. (D) Opening and closing rates of SBD2 versus the glutamine concentration. The data was fitted by eq S5C (opening rates) and S6C (closing rates) as described in the SI. Experiments were performed at −100 mV in 150 mM NaCl, 15 mM Tris-HCl, pH 7.5 at 24 °C by applying a Bessel low-pass filter with a 2 kHz cutoff and sampled at 10 kHz. A postacquisition Gaussian filter of 100 Hz was then applied.