Fig. 4: TAM catalyzes rapid OMP assembly in vitro.
Urea-denatured OmpA or EspPΔ5’ was incubated with 2 μM TAM/PLE proteoliposomes at 30 °C for up to 60 min. Samples were collected at various time points, mixed with loading buffer, and placed on ice. Unheated proteins were resolved by SDS-PAGE, and OmpA was detected by Western blot using an antiserum raised against a C-terminal peptide. The percentage of folded OmpA was calculated using the formula (folded OmpA/total OmpA) x 100 where folded OmpA was defined as protein that migrated faster than the expected molecular weight. EspPΔ5’ samples were mixed with loading buffer, heated to 95 °C, resolved by SDS-PAGE, and detected by Western blot using an antiserum against a C-terminal peptide. Folding was assessed by determining the percent of the protein that underwent proteolytic maturation by using the formula (cleaved β barrel/(cleaved β barrel + uncleaved EspPΔ5’) x 100). Representative experiments are shown at the top. The curves on the bottom show the best fit to the data obtained in five independent experiments performed with OmpA and four performed with EspPΔ5’. The data are presented as mean values +/- standard error of the mean. The rate constant (K) and the time required to reach 50% maximal folding (t1/2) were calculated using GraphPad Prism 8 software.
