Single channel planar bilayer recording experiments

Single channel recording is similar as described before.⁶⁰ Briefly, a ClyA nanopore was inserted into a DPhPC lipid bilayer separating two chambers. Both chambers were filled with 900 μL buffer (150mM NaCl, 15 mM Tris-HCl, pH 7.5). ClyA was added from the cis chamber and inserted into the bilayer spontaneously. MBP was also added from the same chamber to be trapped in nanopore. −80 mV potential was applied across the bilayer and ionic current through nanopore was monitored in voltage-clamp mode by integrated patch clamp amplifier (Axopatch 200B, Molecular Devices). The signal was acquired by an analog-to-digital converter (Digidata 1440A, Molecular Devices) at a sampling rate of 5 kHz after processing with a 4-pole lowpass Bessel filter at 2 kHz. Data was recorded by Clampex software (Molecular Devices). Experiments were conducted at 23 °C.

For data analysis, all traces were filtered with a 50 Hz Gaussian filter. An in-house R script was developed to capture the state transitions of the current traces. Multi Gaussian fitting (mixtools in R) was then applied to fit the all-points histogram, and the peak value of current distribution was then determined. Different bound/unbound states were defined based on boundary of each peak, and assigned back to the original current value. Some current values overlapped between peak boundaries, and the treatment was to reassign the state to the previous state. The transition time of each state was then collected. Cumulated histograms of the transition time of the three bound states to the unbound state were used to calculate the mean first passage time as the average dwell time for each state.