B12-ABP Labeling and CNB12 Competition Studies of Pure Proteins for Fluorescence Gel Analysis, and HL-48 Proteins for LC-MS Proteomic Analyses.

Proteins MetE, the B₁₂-binding domain of MetH, PhrR, and FolD were recombinantly expressed and purified, then the proteins (2 μM in PBS buffer) were labeled with B₁₂-ABP (2 μM) for 30 min at 30 °C. Additionally, competition experiments were performed by carrying out protein labeling with the probe with simultaneous addition of excess CNB₁₂ (10×, 25×, and 50× concentrations versus the B₁₂-ABP). After the 30-min incubation, the samples were UV-irradiated for 10 min at 365 nm. Azido-tetramethylrhodamine fluorophore (2.65 µM) was added to probe-labeled protein solutions followed by the addition of Tris(2-carboxyethyl)phosphine (22 µM), TBTA (Tris[(1-benzyl-1H-1,2,3-traizol-4-yl)methyl])amine) in a 4:1 solution t-butanol:DMSO (44.8 µM), copper sulfate (45 µM), and proteins were separated using 10% (wt/vol) Tris-glycine SDS/PAGE gels. Fluorescence imaging was performed on a Protein Simple FluorchemQ system; the results are shown in Fig. S2.

For HL-48, proteins were labeled as described in the methods, and for CNB₁₂ competition studies simultaneous addition of excess CNB₁₂ (50× concentrations vs. the B₁₂-ABP) was performed before UV irradiation and click chemistry to azido-biotin (see concentrations for click chemistry reagents listed just above). See Materials and Methods for additional details.