Fig. 3: Employing transmembrane protein rotaxanes to analyze protein refolding.

a Experimental configuration. Once the rotaxane has been formed (level 5′), the protein is moved from the trans to the cis side by applying a negative potential (level 6′→level 7′). The retro-translocated substrate remains in level 7′ for a specified refolding time, which is computer controlled. Subsequently, the potential is switched to +100 mV, and the co-translocational unfolding of the refolded protein is recorded (level 2′→3′→4′→5′). This process is then repeated. b Ionic current signal from three cycles as described in ‘a’ with a refolding time of 10 s. c The fraction of cycles in which the refolded substrate shows both levels 2′ and 3′, characteristic of refolding to a native-like state, increases with refolding time. The increase of the folded fraction fits a single exponential (red) yielding a rate constant of 2.5 ± 0.4 s⁻¹. Each point represents data from ~300 translocation events acquired with three different pores. d Frequency histogram of the dwell times in level 2′ observed during 345 cycles with a refolding time of 10 s (data collected with three different pores). The data are well described by an exponentially-distributed population of dwell times (red solid line, k_2’→3’ = 145 ± 10 s⁻¹; black dashed line: probability density function for the native state, k_2→3 = 6.9 ± 0.5 s⁻¹).