Development of antibody fragments that recognize YiiP. (A) Screening was based on phage display of a large library of Fab constructs against YiiP reconstituted in nanodiscs. Results from ELISA assay for several candidates show preferential binding to YiiP embedded in nanodiscs compared with empty nanodiscs, labeled as control. Data represent a single replicate, as is typical for a screen. (B) Formation of a stable complex between detergent-solubilized YiiP and purified Fab candidates was evaluated by SEC. A distinct shift in elution time and a symmetric peak for Fab2 (blue) and Fab3 (purple) is consistent with a stoichiometric complex with YiiP. In contrast, Fab7 (green) produced distinct peaks corresponding to dimeric YiiP and to free Fab, indicating that it did not produce a complex at all. Fab4 (orange) produced smaller shift with an asymmetric peak, probably indicating a nonstoichiometric complex with the YiiP dimer. Profiles for uncomplexed, dimeric YiiP (black), and free Fab (dotted black) are provided for comparison. (C) Transport assays in the presence and absence of Fab2r revealed that complex formation does not affect transport activity of WT YiiP. Fitting of the data (recorded from a single biological preparation in triplicate) with the Michaelis-Menton equation yielded K_M = 1.4 (95% confidence limits, 0.58–3.08) and 1.8 mM (95% confidence limits, 0.83–3.58), V_max = 1.2 (95% confidence limits, 0.95–1.47) and 1.3 (95% confidence limits, 1.04–1.52) s⁻¹ for YiiP and the YiiP/Fab2R complex, respectively. Error bars correspond to SEM. These differences were well within the 95% confidence interval and therefore not statistically significant.