DARPin expression does not significantly alter expression of LmrCD proteins.

<p>(<b>A</b>, <b>B</b>) A V5-tag was introduced in frame at the 5′-end of genomic <i>lmrD</i> in <i>L. lactis</i> (denoted <i>L. lactis NZ9000 lmrD_V5</i>). Plasmid-encoded DARPin activators or the control DARPin E3_5* were expressed in <i>L. lactis NZ9000 lmrD_V5</i> in the presence and absence of daunomycin (14 µM for DARPin_Act3 and E3_5* and 28 µM for DARPin_Act1 and DARPin_Act2, respectively). The expression levels of genomic LmrD_V5 were then quantified by comparing the Western blot signal obtained using an anti-V5 antibody (A) with total protein detected by SYPRO ruby staining (B). (<b>C</b>) The relative amounts of LmrD_V5 expression were quantified by densitometry. Each bar represents the average of three independent data points (n = 3) of which one data point is shown in (A) and (B).</p

ATPase activity of reconstituted LmrCD is stimulated by DARPin activators and daunomycin.

<p>Each symbol or bar represents the average of three data points. (<b>A</b>) The ATPase activity of reconstituted LmrCD is stimulated in the presence of daunomycin in a dose-dependent manner. (<b>B</b>) Reconstituted LmrCD (protein:lipid ratio of 1∶50, proteoliposomes diluted to obtain an LmrCD concentration of 70 nM) was incubated with DARPin activators and control DARPin E3_5* (2.5 µM) and the ATPase activity was determined in the absence and presence of 50 µM daunomycin (triplicates). As a control, buffer instead of DARPins were added to LmrCD. According to t-test analysis, the measured ATPase activity differences between DARPin_Act1 to Act3 and the buffer control are statistically significant (p<0.01 in the absence and p<0.05 in the presence of daunomycin, respectively). (<b>C</b>) The ATPase activities of LmrCD in the presence of DARPin_Act2 and E3_5 were determined over a range of ATP concentrations. The data points were fitted to the Hill equation.</p

Epitope mapping of LmrCD-specific DARPins by ELISA.

<p>(<b>A</b>) Analysis of the LmrCD-specific DARPins by a competition ELISA. Binding of bLmrCD_AviC to immobilized Myc-tagged DARPins was competed with an excess of DARPins devoid of Myc-tag. (<b>B</b>) Schematic drawing of the four proposed binding epitopes on LmrCD recognized by the LmrCD-selective DARPins based on the results of the competition ELISA shown in (A). The number of the epitopes follows the numbering in the main text. (<b>C</b>) The phylogenetic tree of the LmrCD-specific DARPins corresponds well with the proposed binding epitopes. The branches of the phylogenetic tree are highlighted with the color code used to label the four suggested binding epitopes in (B).</p

DARPin binding to membrane-embedded LmrCD.

<p>(<b>A</b>) Six DARPins (each at a 350 nM concentration) specific for AcrB or LmrCD were probed for binding to ISOVs containing either overproduced AcrB_AviC or LmrCD_AviC. Bound DARPins were detected on Western blot (left panel). The signals of the DARPin-specific bands were quantified by densitometry (right panel). Total binding denotes the quantified amount of DARPin bound to membrane vesicles containing overexpressed target protein. Background binding refers to binding to membrane vesicles containing overexpressed LmrCD_AviC in case of the AcrB DARPin 110819, or overexpressed AcrB_AviC when LmrCD-specific DARPins were used. Specific binding was calculated by subtracting background binding from total binding. (<b>B</b>) Binding of DARPin_Act2 and α-LmrCD#3 to ISOVs containing either overproduced AcrB_AviC or LmrCD_AviC was further assessed using increasing concentrations of DARPin (0.35 µM, 1 µM and 2 µM) and analyzed by Western blot (left panel). The data was quantified as in (A) (right panel). The data represent typical results observed in n = 3 experiments.</p

Biophysical characterization of the DARPin-LmrCD complexes.

<p>(<b>A</b>, <b>B</b>) Stoichiometry analysis as exemplified by the LmrCD/α-LmrCD#2 complex. (A) LmrCD and the LmrCD/α-LmrCD#2 complex were separated by SEC (Superdex 200 PC3.2/30, GE Healthcare) with a void volume V₀ = 0.85 ml and a total volume V_t  = 2.4 ml. A fraction corresponding to heterodimeric LmrCD in complex with α-LmrCD#2 complex (red bar) was subjected to protein chip analysis (lane 3, inset). LmrCD and the DARPin α-LmrCD#2 were also analyzed (lanes 1 and 2, inset). The peak at a retention volume of 1.2 ml corresponds to aggregated LmrCD. (B) The peak area of the protein chip chromatogram corresponding to LmrCD and α-LmrCD#2 of lane 3 in (A) were calibrated with dilution series of LmrCD and DARPin of known protein concentrations (not shown) and were used to determine the stoichiometry of the LmrCD-DARPin complexes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037845#pone-0037845-t001" target="_blank">Table 1</a>). (<b>C</b>) Affinities of the DARPins to LmrCD were determined by surface plasmon resonance as shown for α-LmrCD#3. The colored lines correspond to the measured traces at different DARPin concentrations, the fitted curves (1∶1 binding model) are shown as black lines. (<b>D</b>) The steady state DARPin binding signals achieved at the end of the association phase shown in (C) were plotted against the DARPin concentration and fitted using an equilibrium binding equation equivalent to the Michaelis-Menten equation. In this analysis, equilibrium dissociation constants (<i>K</i>_{D, eq.}) were generated.</p

Identification and characterization of DARPin binders by ELISA

<p>(<b>A</b>) Specificity ELISA using bLmrCD_AviC, bMsbA_AviC and bAcrB_AviC as target proteins. Seven DARPins (α-LmrCD#1-5, DARPin_Act2 and DARPin_Act3) were found to be highly specific for bLmrCD_AviC. Many initial DARPin binder-hits promiscuously bound to bLmrCD_AviC, bMsbA_AviC and bAcrB_AviC as exemplified with the “unsp. DARPin” and were therefore not useful for further analysis. DARPins specific for bMsbA_AviC (DARPin_55) and bAcrB_AviC (110819) were used as a positive control. (<b>B</b>) ELISA analyzing binding of the LmrCD-specific DARPins shown in (A) to LmrC (bLmrC-GFP), LmrD (bLmrD-GFP) and the nucleotide binding domain of LmrD (bLmrD-NBD_AviN). Binding to LmrCD (bLmrCD_AviC) was confirmed as positive control.</p