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. Author manuscript; available in PMC: 2021 Mar 25.
Published in final edited form as: J Am Chem Soc. 2020 Mar 11;142(12):5482–5486. doi: 10.1021/jacs.9b12185

Figure 3.

Figure 3.

The captured MurJ×Lipid II species represents an on-pathway intermediate in Lipid II transport. (a) The protonophore CCCP inhibits MurJ cycling. (b) Cells were pre-treated with 100 μM CCCP for 5 min, then diluted 100-fold to wash out CCCP and restore MurJ function. After 0 – 20 min, Lipid II levels were assessed as previously described7. Cells were then UV-irradiated for 5 min and assessed for MurJ×Lipid II formation. (c) The MurJA29C variant is inactivated by the thiol-labeling probe MTSES. This inactivation is reversed by the reducing agent DTT. (d) Cells were pre-treated with 400 μM MTSES for 5 min then diluted 10-fold into media containing 10 mM DTT to restore MurJ function. After 0 – 10 min, Lipid II levels were assessed. Cells were then UV-irradiated for 5 min and assessed for MurJ×Lipid II content. See also Figures S1, S10–S13.