FIGURE 5.

Multiple mutations strongly destabilize the EspP β-helix. A and B, close-up view of two regions of the β-helix showing the residues that were converted to alanine to create mutants S and Q. The illustration was generated using PyMOL. C, AD202 cells transformed with pRLS5 or a plasmid encoding mutant S or Q were grown in LB medium, and expression of the plasmid-borne gene was induced by the addition of IPTG. Cells were removed by centrifugation, and half of the culture medium was treated with PK. Western blot analysis was then conducted using the anti-EspP passenger domain antiserum. D–G, the experiment described for C was repeated except that chymotrypsin or trypsin was added to equal portions of the culture medium at the indicated concentrations. In E and G, only the culture medium of cells transformed with pRLS5 was analyzed.