Fig. 1.

Experimental setup, ATPase rate and fluorescence quenching by ATP and BtuF. a Structure of BtuF (pink) bound to BtuCD (BtuC homodimer: green, BtuD homodimer: blue) in the absence of nucleotide and substrate (Protein Data Bank (PDB) ID 2QI9). Cysteine mutations for labelling in BtuF (D141C) and BtuC (Q111C) are marked with a yellow dot. The distance between the labelling positions is ~37 Å. b ATPase rate of various BtuCD-F mutants with or without fluorescent label reconstituted in liposomes and loaded with (pink bar) or without (grey bar) vitamin B₁₂. BtuCD WT denotes the wild-type, BtuCD_cys denotes the cysteine mutant and BtuCD_EQ denotes the cysteine mutant that is ATPase-impaired. For all combinations, the cysteine mutant of BtuF is used. Measured rates are not corrected for orientation of the transporter. When BtuF is present at the concentrations used, the full complex is formed. Values displayed are the mean and standard deviation of three experiments. c Experimental design (fluorescent labels are omitted). BtuCD was reconstituted in liposomes of 100-nm diameter in ratios such that on average one transporter was found per liposome. By introducing BtuF and vitamin B₁₂ to the lumen of the vesicle and ATP on the outside or vice versa, only one particular orientation of the transporter was probed. Proteoliposomes are tethered to a glass surface via a biotin-streptavidin link and imaged using TIRF microscopy. d A complex of BtuCD_cys labelled with Alexa Fluor 555 and unlabelled BtuF showed decrease in fluorescence intensity upon addition of 2 mM ATP and 10 mM Mg²⁺ on the outside (middle panel). The distribution of event times of the first drop of intensity is plotted in a histogram (right panel) for the positive (pos, with ATP) and negative (neg, without ATP) experiment. For a description of the data analysis, see methods. e Similar experiment as described in c, but with the ATPase impaired mutant BtuCD_EQ. No events were observed. f Similar experiment as described in c, but the vesicle lumen was left empty. Upon introduction of BtuF to the outside of the liposomes an increase in fluorescence intensity was observed. For each condition in d–f around 1000 single-molecule fluorescence traces were analysed