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. 2018 Aug 31;7:e40889. doi: 10.7554/eLife.40889

Figure 2. Ser23 and Pro24 act together to enforce the reentrant PglC topology.

(A) Schematic representation of SCAM analysis used to assess the topology of wild-type PglC and variants. Cyan starbursts (top) indicate the location of unique cysteines introduced into PglC. (B) SCAM analysis of wild-type PglC topology (* = native PglC; ** = PglC labeled with PEG-mal; C = control, no PEG-mal labeling). (C) SCAM analysis of S23A, P24A and S23A/P24A PglC variant topologies. All SCAM experiments were performed in duplicate or more. Representative Western blots are shown.

Figure 2.

Figure 2—figure supplement 1. PglC Cys variants used for SCAM analyses.

Figure 2—figure supplement 1.

Activity assays of wild-type SUMO-PglC and K4C, F6C, S88C and S186C SUMO-PglC using UMP-Glo to monitor UMP release. Assays indicate that PglC variants used for SCAM analyses retain 10–50% of native catalytic activity. Error bars are given for mean ± SD, n = 2.