Fig. 2: AK_2+ current blockades in the ClyA-AS nanopore upon binding of Ap5A, ATP, and AMP.

A Expansion of a typical blockade induced by the apo-AK in ClyA-AS. B A typical Ap5A ligand-induced events showing four additional levels (M1-M4). C Scheme indicating the transitions observed with Ap5A and their molecular interpretation. Typical blockades in the presence of 1 mM ATP (D), 1 mM ATP and 1 mM AMP (E), 1 mM ATP and no Mg²⁺ in solution (F), and 1 mM ATP and 1 mM AMP with no Mg²⁺ (G). H Frequency of LID closing - measured as the inverse of dwell times – at increasing ATP concentrations. The line indicates a linear fit. I LID opening frequency at increasing ATP concentrations. The curves are fits of the data to a Hill function. J Percentage of the closed LID domain at increasing ligand concentrations. The curves correspond to fits of a Hill function. The measurements were performed in 400 mM KCl, 15 mM Tris, 2 mM MgCl₂ (except for panels F and G), pH 7.5 at room temperature (22 °C) applying −90 mV (trans) and sampling at 50 kHz with a 10 kHz Bessel-low filter, additionally digitally filtered with a 2 kHz Gaussian low-pass filter. Error bars represent the standard deviation of the mean between independent experiments (N = 3). Ligands were added to the trans chamber and the enzyme to the cis chamber.