Figure 2: Lifetimes of level 3 for N terminus-first translocation.

(a) Event histogram of the dwell times in level 2 (step 2→3) for Trx V5-C109-oligo(dC)₃₀ at +140 mV (ref. 11). The fit is to a single exponential function and yields a rate constant k_2→3=70 s⁻¹. (b) Event histogram of the dwell times in level 3 (step 3→4) for Trx V5-C109-oligo(dC)₃₀ at +140 mV (ref. 11). The fit is to a single exponential function and yields a rate constant k_3→4=1.7 s⁻¹. (c) Free energy profile of the unfolding steps (2→3 and 3→4) for C terminus-first translocation at +140 mV. A pre-exponential factor of 10⁻⁸ s⁻¹, and ΔG(N→U)=−90 kJ mol⁻¹ and ΔG(N→I)=−55 kJ mol⁻¹ were assumed, where: N, folded protein; U, unfolded protein; I, unfolding intermediate. (d) Event histogram of the dwell times in level 2 (step 2→3) for Trx oligo(dC)₃₀-S1C-V5 at +140 mV. The fit is to a single exponential function and yields a rate constant k_3→4=690 s⁻¹. (e) Event histogram of the dwell times in level 3 (step 3→4) for Trx oligo(dC)₃₀-S1C-V5 at +140 mV. The fit is to a double exponential function and yields rate constants k_3a→4=0.47 s⁻¹ and k_3b→4=0.0087, s⁻¹. The dashed line is the deconvolution into single exponentials. (f) Free energy profile of the unfolding steps (2→3 and 3→4) for N terminus-first translocation at +140 mV. A pre-exponential factor of 10⁻⁸ s⁻¹, and ΔG(N→U)=−90 kJ mol⁻¹, ΔG(N→I_a)=−45 kJ mol⁻¹ and ΔG(N→I_b)=−50 kJ mol⁻¹ were assumed, where: I_a and I_b are the two unfolding intermediates. Data for C terminus first are from ref. 11. Data were collected on six different pores in the case of C terminus first and on 17 different pores in the case of N terminus first.