Fig. 4.

Transport of vitamin B₁₂ observed with fluorescence quenching. a Dye quenching by vitamin B₁₂. His-tagged BtuF labelled with Alexa Fluor 488 was immobilised on the glass surface via anti-His antibodies. Upon introduction of substrate, the fluorescence was quenched (middle panel) before it bleached around 100 s. Fluorescence intensities were collected in a histogram (right panel) to show that the quenching effect was ~75%. b When unlabelled BtuCD was reconstituted in liposomes loaded with 10 µM vitamin B₁₂ (~3 molecules) and BtuF labelled with Alexa Fluor 488, a decrease in intensity was observed upon introduction of ATP and Mg²⁺ (middle panel). The orange line marks the periods of low fluorescence and indicates when a substrate molecule was present in the transporter. Averaging all traces (right panel) shows a gradual increase in intensity, which we attribute to depletion of substrate from the liposome. For data analysis, see methods. c Similar experiment as described in b, but with ten times higher concentration of vitamin B₁₂. Depletion as observed in b was not visible here. For each condition, more than roughly 1000 traces were analysed