Figure 2
Truncation of membrane-extrinsic domain abrogates QC activity. (a) Schematic representation of the α3 family of designed three-helix-bundle proteins developed by DeGrado and coworkers32,33 which represent a continuum of folded structures, ranging from aggregation-prone (α3A) and monomeric molten globular (α3B) to native-like, well-ordered (α3C and α3D). (b) Resistance of serially diluted DADE cells co-expressing spTorA-α3-Bla chimeras (A, B, C or D) along with a Tat operon plasmid encoding either wt TatB or one of the TatB variants. Cells were spotted on LB-agar plates containing either 100 μg/mL Amp or 25 μg/mL chloramphenicol (0 μg/mL Amp). Dashed lines denote different LB-agar plates that were all generated and imaged at the same time. (c) Western blot analysis of cytoplasmic and periplasmic fractions prepared from DADE cells co-expressing Tat-targeted PhoA from pTorA-AP along with a Tat operon plasmid encoding either wt, one of the TatB variants, or mut51. An equivalent number of cells was loaded in each lane. PhoA was probed with anti-PhoA antibody while anti-GroEL antibody confirmed equivalent loading in each lane. Asterisk indicates spTorA-PhoA breakdown product. Molecular weight (MW) markers are indicated on the left. Results are representative of three biological replicates. See Supplementary Fig. 1 for uncropped versions of the images.
