Fig. 1.
LptD is fully oxidized but disulfide bonds are not required for LptD stability. (A) Whole-cell samples from wild type (wt, NR754) and ΔsurA (NR1215) were boiled in the absence or presence of β-ME and subjected to LptD immunoblot analysis. Reduced and oxidized LptD (LptDRED and LptDOX, respectively) and an unidentified 55-kDa IM protein are marked (23). Size (in kilodaltons) of molecular mass markers are shown on the left. (B) Samples from NR754 (pET2342lptDSSSS), which produces LptDCCCC (marked as CCCC) and LptDSSSS (marked as SSSS), were subjected to nonreducing SDS/PAGE and immunoblotting using anti-LptD antiserum. (C) Samples from NR754 ΔlptD (pET2342lptDCCCC), NR754 ΔlptD (pET2342lptDSCSC), and NR754 ΔlptD (pET2342lptDCSCS) were analyzed by nonreducing SDS/PAGE followed by LptD immunoblotting. Different LptD proteins adopt different shapes (Right) when denatured because of the connectivity of their disulfide bonds. The double and solid lines represent the N- and C-terminal domains of LptD, respectively.