Figure 2.

Effect of nanobodies on BtuCD-F function. (A) Schematic of the substrate-binding assay. Fluorescently labeled BtuF (BtuF_fluo) was used to measure cyanocobalamin (Cbl) binding in the presence of nanobody. (B) Equilibrium Cbl binding to BtuF_fluo. Shown is the normalized fluorescence signal against substrate concentration (the raw fluorescence data is shown in Supplementary Figure 1). 5 nM BtuF_fluo, Cbl concentrations ranging from 0.3 nM to 10 μM, and different Nb concentrations were used (5 μM for Nb1 and Nb14; 1 μM for Nb7, Nb15 and Nb17; 100 nM for Nb9 and Nb10). Affinity values for nanobody-BtuF binding were determined by numerical evaluation of the competitive binding data and shown in Table 1. Note that Nb1 is a control nanobody that does not bind BtuF. C) Schematic of the spheroplast-based substrate transport and BtuF_fluo binding assays.⁵⁷Co-cyanocobalamin (⁵⁷Co-Cbl) transport into spheroplasts overexpressing WT BtuCD was measured in the presence of Nbs. (D) The BtuCD expression level in the spheroplasts was determined by the amount of BtuF_fluo associated with the spheroplasts. Cells transformed with a plasmid containing WT BtuCD but without expression induction (‘WT uninduced’) served as a control. The fluorescence was detected using excitation at 485 nm and emission at 516 nm. (E) Cbl transport in the presence of Nbs. The following concentrations were used: 5 μM BtuF, 15 μM Cbl, 75 μM nanobodies and 0.08 g/ml spheroplasts (~0.45 μM BtuCD). A hydrolysis-deficient BtuD mutant, E159Q, was used as a negative control. Shown are mean and SEM of the transport rates determined by linear regression using 5 time points.