Figure 5: OM tension accelerates transmembrane β-barrel folding.

(A) Hypothetically unraveled EspP β-barrel with the position of the conserved β-signal strand (green) held constant to its angular membrane orientation when fully folded (58° for EspP, membrane plane calculated using OPM server) (Lomize et al., 2012) showing intrinsic mismatch between the membrane plane and the transmembrane portion of β-strands (red). Loops and turns are not drawn to scale. (B) EspP β-sheet as in A except model depicted with the experimentally determined angle (using the BAM-^MBP−76EspP structure) of EspP β-signal relative to the membrane when hybridized to BamAβ1 (cyan). The model predicts that the intrinsic tension forces of the rigid OM aids β-barrel folding by countering the intrinsic deflection of an incompletely folded expanded β-sheet. (C) E. coli expressing ^HisBamABCDE and ^MBP−76EspP were suspended in LB (isotonic control) or LB-sorbitol (hypertonic). β-barrel folding was restarted from the hybrid-barrel stage by adding PK to release a C-terminal EspP fragment (frags^(C)). Fragment conversion to a completely folded β-barrel after PK addition was monitored by immunoblotting with an antiserum against the C-terminus of EspP (αEspP_βC). Left, representative blots. Right, mean fraction of the EspP PK fragment (top) or converted into a folded β-barrel (bottom) (±SEM, n=4). (D) E. coli expressing ^HisBamA_S425CBCDE and ^MBP−76EspP_S1299C were grown and treated as in C except that samples at 5 min post-PK were mock treated (Ox-) or treated with 4-DPS (Ox+) and ^HisBamA•^MBP−76EspP crosslinks were identified by double-immunoblotting with αEspP_βC and αHis antibodies (n = 3). Non-specific crosslinks are denoted (<). Right, fraction of crosslinked BamA•^MBP−76EspP C-terminal PK fragment detected with αEspP_βC [line at median, two-tailed paired t-test: P=0.0038 (**)]. (E) Bacteria were treated as in C except that after 5 min of PK treatment the media was exchanged for either LB-sorbitol (hypertonic) or LB (hypotonic) (gray plot area). Plots are mean fraction (±SEM, n=3). For C and E, 2-way repeated measures ANOVA (Šídák’s tests) were performed *p < 0.05, **p < 0.01, and ****p < 0.0001.