Figure 1.

Immobilization of AcrB. (A) Purification and biotinylation of AcrB^S1043C. Purified AcrB^S1043C (0.5 μg) and its biotinylated derivative Cys1043^bio were mixed with the SDS-sample buffer plus/minus 10 mM DTT and separated by 10% SDS-PAGE. Protein was either stained with Coomassie Brilliant Blue (CBB) or transferred onto PVDF membrane and incubated with streptavidin alkaline phosphatase (anti-biotin). Cys1043^bio–anti-biotin complex was visualized with nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) salt substrates. (B) Fractionation of Cys1043^bio by size exclusion chromatography. Cys1043^bio was injected onto YM-Pack Diol-300 column at 0.5 ml/min in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.03% DDM (Tris buffer). Eluted fractions (0.25 ml) were collected, of which B11 and B14 fractions were used in SPR assays. (C) Anti-biotin Western analysis of Cys1043^bio (2.3 μM) treated with indicated concentrations of the amine-reactive cross-linker dithiobis[succinimidyl propionate (DSP) in buffer containing 20 mM Hepes-KOH (7.7), 150 mM NaCl and 0.03% DDM. The reactions were carried out for 30 min at 37°C. Monomeric AcrB and its cross-linked dimers and trimers are indicated. (D) Kinetic analysis of DARPin binding to AcrB. Two-fold serial dilutions of DARPin (12.5–800 nM) in Tris buffer were injected over Cys1043^bio (AcrB thereafter) immobilized at density 2040 RU (red lines). Data were fit globally to a 1:1 Langmuir model (black), giving k₁=1.03×10⁶ M⁻¹s⁻¹, k₋₁=0.047 s⁻¹, leading to K_D=46 nM. (D) DARPin interaction with AcrB fractions. The unfractionated Cys1043^bio (B total) and its B11 and B14 fractions were immobilized at densities 2384 RU, 2348 RU and 2414 RU, respectively. DARPin (100 nM) was injected at 50 μl/min for 2 min in running buffer containing 20 mM MES-KOH (pH 6.0), 150 mM NaCl, 0.03% DDM (MES buffer). See also Table 1S.