Substituted-cysteine accessibility method (SCAM)

PglC from C. jejuni strain 11168 was used for SCAM analysis¹⁵. Unique cysteines were introduced either N-terminally (K4C, F6C) or C-terminally (S88C, S186C) to the membrane-associated domain at non-conserved, surface-exposed sites (residues correspond to N4, L6, S89, and S187 in PglC from C. concisus) (Supplementary Table 5). Wild-type PglC and the four cysteine variants were overexpressed in E. coli. Whole cells expressing each unique variant were treated with one of two thiol-blocking reagents, either N-ethylmaleimide (NEM), which is cell-permeant, or 2-sulfonatoethyl methanethiosulfonate (MTSES), which is only able to cross the outer cell membrane. Following cell lysis, any remaining free cysteines were reacted with PEG-maleimide (PEG-mal). Labeling of the target protein with PEG-mal was observed by Western blot as a band shift to higher molecular weight. Cysteines in the periplasm are thus distinguished by their ability to be blocked from PEGylation by both NEM and MTSES, while cytoplasmic cysteines are PEGylated following treatment with MTSES but not following treatment with NEM. Wild-type PglC has no native cysteines, and thus was not labeled with PEG-mal under any thiol-blocking conditions. All four cysteine variants were blocked from PEG-mal labeling by incubation with NEM but not by incubation with MTSES, indicating that all four are located in the cytoplasm (Fig. 2b). A unique cysteine variant of a periplasmic protein, PEB3 A204C, served as a positive control for thiol-blocking of cysteines in the periplasm.