Fig. 6: InhA and TGF-β interact with partially overlapping, but distinct regions of BGZP-C.
a–c Isoleucine δ1 methyl region of the 1H−13C correlation NMR spectra of BGZP-C (black). Upon TGF-β2 (a) or mmTGF-β2 (b) binding (red), the chemical shift of I161 δ1 methyl is perturbed, with the signal shifting from the random coil to the structured region, consistent with a disordered loop to helix transition. No shifts were observed upon binding of InhA (c). Representative measurement of signal intensity (d) and line width (e) changes of methyl signals upon InhA titration into the Ile δ1, Met, Val and Leu methyl-labeled BGZP-C sample. Each dot represents a line width of a single residue upon titration. Mean value of signal intensity or line width within a sample is represented by a red bar. The measurement was repeated twice. f Confocal microscopy images of competition assay - HEK293T cells stably transfected with a SNAP-tagged BG construct (SNAP-BG) stained with DAPI (blue, left panels), incubated with a SNAP-tag ligand conjugated to the CF567 fluorescent dye (red, middle panels), along with InhA tagged with CF640R fluorescent dye (violet, right panels) alone (row 1) or with the addition of the indicated unlabeled ligands (rows 2–5). Bound InhA could be outcompeted by addition of excess unlabeled InhA, TGF-β2 or mmTGF-β2. Scale bar, 50 µm. g Quantification of the fluorescent colocalized SNAP-BG/InhA signals. Each data point represents the ratio of SNAP-BG signal to InhA signal within a segmented region of the image generated by applying the Otsu thresholding method to the SNAP-BG channel. Analysis was performed on three images per condition, each corresponding to an independent biological replicate (n = 3). h Chart summarizing SPR binding experiments using selected panels of BGZP-C mutants binding to immobilized TGF-β2 or InhA. Depicted mean KD values with standard deviation were derived from series of dilution in three consecutive repeats. i Selected SPR binding sensorgrams show response to a 2-fold dilution series of injections of BGZP-C variants (black) over TGF-β2 or InhA immobilized on the chip surface. Fitting curves (red) were generated based on three consecutive repeats. Source data are provided as a Source Data file.
