Fig. 2
Prediction and experimental validation of membrane integration of TMS of T3SS substrates a Distribution of the calculated membrane integration propensity (ΔGapp) of TMS of T3SS substrates (red) compared to previously published values for ΔGapp of regular transmembrane- and soluble proteins, respectively20. For each protein, only its lowest ΔGapp of any given sequence window is shown (ΔG predictor settings: window size: 18–35 aa, length correction: ON). b Principle of the TMS insertion assay, see results and methods sections for description21. c Relative membrane insertion of the indicated TMS was analyzed in E. coli BW25113 with the TMS insertion assay. ± ara indicates induction of expression of the respective test construct. The fate of the Lep-LacY chimera was assessed by SDS PAGE, Western blotting and immunodetection of the Lep P2 domain. A representative result of three independent experiments is shown. The fraction of inserted Lep-LacY (fI) was calculated from the ratio of uncleaved to cleaved (indicated by asterisk) Lep-LacY, corrected for different degradation rates of the individual fragments according to Öjemalm et al21.. Abbreviations: ara: arabinose; Lep: leader peptidase; Lep-LacY (T+I): translocated and integrated membrane helix; Lep-LacY (Tsmall): small translocated form of Lep-LacY. IM inner membrane, TMS transmembrane segment
