Fig. 1.

Overlay of the representative SEC of purified WT CsgE (red trace) and W48A/F79A double mutant (blue trace). Black arrows mark the void and total retention volume of the column, determined using standard markers. The retention volume of the double-mutant W48A/F79A corresponds to a SEC-molecular weight about 13 kDa, according to the standard globular molecules (Fig. S1), consistent with the native spray mass spectrometry measurement (Fig. S2). However, purified WT CsgE partitioned into two distinct peaks (marked with red arrow and *), although it showed a single band on SDS/PAGE (Inset). The retention volume of the main peak (∼90%, red*) is greater than the total retention volume. The measured mass of this main peak (red*) by native spray mass spectrometry (Fig. S2) is about 13,212 Da, confirming it is the monomeric WT molecule. The oligomeric peak of WT (red arrow) could not be measured by our native spray mass spectrometry. The approximate ratio of the SEC molecular weight for the main (red*) and minor (red arrow) peaks was about 1:9 (Fig. S1), suggesting the minor peak (red arrow) of WT on SEC is a nonamer, consistent with the previous report (37). On SDS- and native-PAGE (Inset), lane # was the same sample as loaded in SEC analysis; lane * was the peak marked * in SEC for WT CsgE and W48A/F79A CsgE.