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Fig. 3 (a) Overview of truncated versions of 24fα. The positions of the cross-linking amino acids (X, light gray) and of the modifier-bearing amino acid (m, red) are indicated (for peptide details, see Table S3). (b) Growth assay using E. colilptD4213 (imp) after treatment with 24fα, 20fα, 17fα, and 15fα (c = 25 μM). The optical density at 600 nm (OD600) was measured every 15 min over 22 h. Measurements were conducted in triplicate (n = 3 technical replicates, error bars = SD). (c) Growth assay using E. colilptD4213 (imp) after treatment with 17fα–δ (c = 50 μM). The optical density at 600 nm (OD600) was measured every 15 min over 22 h. Measurements were conducted in triplicate (n = 3 technical replicates, error bars = SD). (d) 17% Tris/Tricine PAGE (protein modification assay) assessing peptide binding to FtsQ(50–276). Covalent inhibitors 17fα–δ (c = 125 μM; for peptide details, see Table S3) were incubated with FtsQ(50–276) (c = 50 μM) for 1 or 3 h. Up-shifted bands are indicative of modified FtsQ. (e) MS of FtsQ(50–276) after 17fα treatment. Signals corresponding to unmodified (gray) and 17fα-labeled FtsQ(50–276) are indicated. Obtained masses (obtained by deconvolution) are shown (calculated masses, FtsQ: 26939 g/mol, FtsQ-17fα: 29061 g/mol). The obtained mass difference (ΔMW = 2124 g/mol) corresponds well with the one expected after a reaction with 1 equiv of 17fα (ΔMW = 2122 g/mol; for details, see Figure S9). (f) Concentration-dependent effect of 17fα and 15fα (c = 0.78 μM–2100 μM) on the growth of E. colilptD4213 (imp) after 15 h. All measurements were performed in triplicate (n = 3 replicates, error bars = SD).