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. Author manuscript; available in PMC: 2011 Apr 27.
Published in final edited form as: Mol Microbiol. 2009 Apr 30;72(5):1171–1180. doi: 10.1111/j.1365-2958.2009.06715.x

Figure 1. Accessibility of Cys residues in FepA to FM-labeling at 0 °C and 37 °C, and during ColB binding (at 0 °C) and uptake (at 37 °C).

Figure 1

OKN3 (fepA) harboring plasmids carrying fepA+ or its derivatives that encode Cys substitutions mutations were prepared as described in Experimental Procedures, resuspended in PBS, pH 6.5, and incubated for 30 min at 0 °C or 37 °C in the absence or presence of ColB, and then exposed to FM (5 uM) for 15 min at the same temperature. Cells lysates were resolved by SDS-PAGE and fluorescence images from the gels (Fig. S2) were analyzed by IMAGEQUANT (Molecular Dynamics). Each panel shows the mean FM-labeling of FepA (relative to band 3) from three or more independent experiments, with associated standard error. A. 0 °C vs 37 °C. White bars derive from cells labeled at 0 °C; yellow bars are from cells labeled at 37 °C. The inset shows fluoresceination of BSA at 0 °C, 25 °C and 27 °C, in the presence of 5 uM (grey bars) and 50 uM (black) FM. B. 0 °C, ± ColB. At 0 °C the cells are metabolically inactive, so ColB binds but is not transported. White bars derive from cells labeled at 0 °C in the absence of ColB; light blue bars are from cells labeled at 0 °C in the presence of ColB. C. 37 °C ± ColB. At 37 °C the cells are metabolically active, so ColB binds and kills. Yellow bars derive from cells labeled at 37 °C in the absence of ColB; green bars are from cells labeled at 37 °C in the presence of ColB. D. 0 °C vs 37 °C, + ColB. The graph compares the effects of ColB on the labeling of FepA Cys mutants at 0 °C and 37 °C. No increases in FM-labeling of N-domain residues were observed during ColB killing.