Fig. 3. Interaction of SR1 with membrane-bound αHL-GroES_L monitored by single-molecule recordings.

(A to C) Current blockades provoked by SR1 to αHL-GroES_L in the presence of 1 mM ATP (A), the nonhydrolyzable ATP analog AMP-PNP (B), or adenosine diphosphate (ADP) (C) added to the cis chamber at +100 mV. The current blockades are due to the interaction of the chaperonin with individual engineered co-chaperonin nanopores. (A) Fifty percent of the blockades in the presence of ATP were transient (dwell time, 55 ± 5 s; main I_B value, 5.2 ± 0.2 pA), showing fast current fluctuations (see the text and fig. S5). The remaining current blockades switched to a quieter and permanent current level (I_B value, 12.5 ± 0.5 pA). The open pore current could only be obtained by multiple ramping of the potential to +100/−100 mV (red asterisks). (B) AMP-PNP–induced current blockades showed the same current level as ATP-induced blockades but were always transient (average dwell time, 157 ± 64 s; main I_B value, 5.2 ± 0.1 pA) (see the text). (C) In the presence of ADP, the current blockades were transient, showing a dwell time faster than the resolution of our current recordings (dwell time, <1 ms). Occasionally longer current blockades were observed, which showed an I_B value of 3.0 ± 1.4 pA (fig. S6c). The electrical recordings were carried out in 1 M KCl, 50 mM tris-HCl (pH 7.5), and 5 mM MgCl₂ at 23°C and +100 mV by applying a 10-kHz low-pass Bessel filter with a 50-kHz sampling rate. An additional digital Gaussian filter at 2 kHz was applied to current traces.