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. 2013 Jul 16;105(2):356–364. doi: 10.1016/j.bpj.2013.04.046

Figure 3.

Figure 3

Reversible and sequential binding of two Ag+ ions to a single cysteine residue. (A) Current recordings at different free Ag+ ion concentrations, [Ag+]free (Ag+ was added as AgNO3 to the cis compartment). Each current level is labeled on the right: PC = unoccupied pore (level 0); PCS-Ag = one Ag+ ion-bound (level 1); PCS+(Ag)2 = two Ag+ ions-bound (level 2). All-points amplitude histograms are shown on the left. Conditions: 2 M KNO3, 10 mM MOPS, 10 mM EDTA, pH 7.4, at 22°C and –50 mV. Under these conditions, PC has an open pore current of –96 ± 2 pA (n = 5). (B) Dwell-time histograms for PC, PCS-Ag, and PCS+(Ag)2 in the presence of 66 nM [Ag+]free. Each histogram was fitted to a single-exponential function using Clampfit (Molecular Devices) to determine the mean dwell time (τ¯). (C) Plots of the measured rates of each transition versus [Ag+]free. Each data point (mean ± SD) was obtained from three experiments: level 0→level 1 (blue solid rhombus), level 1→0 (blue open rhombus), level 1→2 (red solid triangle), level 2→1 (red open triangle). The determination of rate constants from these plots is described in the Materials and Methods section. (D) Kinetic scheme describing the sequential association of two Ag+ ions with the cysteine side chain in PC. Direct transitions between PC and PCS+(Ag)2 are not observed. This scheme was used in QuB (see Materials and Methods) for the determination of rate constants.