Fig. 1: Adenylate kinase inside the ClyA-AS nanopore.

A Crystal structure of E. coli adenylate kinase in open (PDB: 4ake) and closed conformation (Ap5A bound, PDB: 1ake). The enzyme is composed of a rigid core domain (CORE, grey) and two flexible domains that are closing upon binding of ATP, the LID domain (red, residues 121–161), and AMP, the NMP domain (blue, residues 3–60), respectively. B Residues within 6 Å from Mg²⁺ in the closed AK bound to Ap5A (the coordinates for Mg²⁺ were taken from PDB: 5G3Z). The dotted black lines indicate a binding distance of <3 Å, the dotted grey lines a distance >4 Å. The interactions between the three domains are all mediated by Ap5A. Mg²⁺ is at binding distance only with the phosphate groups of Ap5A. C Schematic representation of AK, extended with two additional lysine residues and a Strep-tag at the C-terminus (AK_2 + )(Sequence depicted below), inside a ClyA-AS nanopore in a lipid bilayer (grey area). D Typical current trace after addition of 100 nM AK_2+ to a single ClyAS-AS nanopore in cis at −90 mV applied potential (trans). The grey line shows the open-pore current (I_o, −433.3 ± 5.7 pA), the black dashed line (M Type-a, −200.3 ± 2.4 pA) and the grey dashed line (M Type-b, −249.9 ± 3.7 pA) represent the blocked pore current. Error represents the standard deviation of the mean between independent experiments (N = 3). The measurements were performed in 400 mM KCl, 15 mM Tris, 2 mM MgCl₂, 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.