Extended Data Fig. 9: Disruption of folded and aggregated structures by ClpB.

a, Extension length (L_e) of the 4MBP construct plotted against time in the presence of ClpB(Y503D) and ATP. b, Cartoons of event sequence suggested in a. (1) One MBP core is unfolded by increasing the force, immediately followed by relaxation to 5 pN to avoid unfolding other MBP cores. Some C-terminal helices also unfolded in this process. (2) After a waiting period, ClpB binds the unfolded part and translocates it completely. (3) ClpB reaches the neighbouring folded MBP domain (and the DNA tether), and hence no longer changes L_e. (4) After a short pause, L_e increases in a discrete step of 270 aa, indicating the unfolding of one MBP core, which has precisely that length. (5) ClpB(Y503D) translocates briefly immediately afterwards, further indicating the bound ClpB, and (6) back-slipping occurs. Note that the length of the unfolded chain has increased by 270 aa, the length of one MBP core, as expected (star). (7) Translocation continues. c, d, To create a misfolded or aggregated state, the 4MBP construct was unfolded and rapidly relaxed (green trace). This sometimes produced non-native structures characterized by being compact and highly resistant to force (red trace). The tether was then relaxed to low force. e, Subsequent measurement of extension length against time. f, Cartoons of event sequence suggested in e: (1) the length remains unchanged, for example, owing to waiting for ClpB binding. (2) The length increases abruptly by about 600 aa, which is more than one MBP core (270 aa), suggesting that ClpB disrupted a non-native (aggregated) structure that contained more than one MBP repeat. (3) ClpB translocation is observed immediately afterwards. This is consistent with the model, because one-step disruption of structures by ClpB (pushing) action can yield unfolded polypeptide segments directly on the cis side of ClpB that are then available for translocation. Note that polypeptide may also be liberated on the other side of the misfolded structure, which is not immediately available for translocation. Subsequently, further translocation and slipping behaviour is observed. Note that the structure becomes almost fully disrupted, as it nears the maximum length of 1,440 aa.