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. 2018 Feb 9;4(2):eaaq0243. doi: 10.1126/sciadv.aaq0243

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Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

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Fig. 1 — (A) Scheme of the distinct protein conformations visited by ubiquitin [Protein Data Bank (PDB): 1UBQ] when exposed to mechanical force, namely, native, collapsed, and extended (22). (B) Schematics of the single-molecule nanomechanical experiment, whereby a ubiquitin polyprotein (Ubi)₉ is tethered between a gold substrate and an atomic force microscopy cantilever tip. (C) A force-quench protocol demonstrates that ubiquitin can quantitatively refold in t_q = 5 s, marked by the presence of 20-nm steps in the test pulse. (D) By contrast, upon addition of 5 μM DnaJ, the refolding efficiency is significantly decreased. (E) The kinetics of ubiquitin refolding is measured by changing t_q, spanning the range 0.5 to 15 s (blue symbols; t_q = 0.5 s, n = 37 individual trajectories; t_q = 1 s, n = 56; t_q = 2 s, n = 36; t_q = 5s, n = 46; t_q = 10 s, n = 44; t_q = 15 s, n = 26). A single exponential fit to the refolding kinetics of wild-type ubiquitin displays a folding rate k_f = 0.52 ± 0.05 s⁻¹. In the presence of DnaJ (green symbols), the refolding rate is decreased down to k_f = 0.29 ± 0.07 s⁻¹ (t_q = 0.5 s, n = 36 trajectories; t_q = 1 s, n = 30; t_q = 2 s, n = 39; t_q = 5 s, n = 37; t_q = 10 s, n = 35; t_q = 15 s, n = 52). Such a reduced refolding rate can be explained in terms of the delayed ubiquitin collapse in the presence of DnaJ (fig. S1). (F) Whereas ubiquitin reaches a high refolding yield (~65%), the addition of DnaJ (green symbols) significantly (P < 0.0001) reduces the yield down to ~30% at a quench time of t_q = 5 s. (G) The unfolding kinetics of ubiquitin at 120 pN in the absence and presence of 5 μM DnaJ does not result in a change in the unfolding rate [k_u = 0.93 ± 0.06 s⁻¹ (n = 195 unfolding trajectories) and k_u = 0.94 ± 0.07 s⁻¹ (n = 138 unfolding trajectories), respectively], suggesting that DnaJ does not bind to the native state of ubiquitin. (H) These results are confirmed with pull-down assays, demonstrating that DnaJ and the folded ubiquitin do not coprecipitate (P, pellet; S, supernatant).