FIG 7 .

OprF mutants with higher porin activity contain larger fractions of one-domain conformers. The fraction of the one-domain conformer was assessed by determining the extent of maleimide-PEO₂-biotin labeling of the A312C residue of OprF in intact cells. Intact cells were labeled with maleimide-PEO₂-biotin (10 to 100 µM) for 5 min at room temperature. After the reaction was stopped by the addition of 2-mercaptoethanol, proteins from identical numbers of cells were separated by SDS-PAGE, and the blot was stained with streptavidin reagent for biotin as described in Materials and Methods. The enhanced chemiluminescence (ECL) profile was recorded (B), and the intensity of maleimide-PEO-biotin bound to OprF was quantified after each band was scanned by using NIH Image software (A). Panel B shows the labeling with (from left to right) 20, 40, 60, 80, and 100 µM reagent for the indicated strains.