Fig. 4: In vitro characterization of OptoNB binding.
From: Optogenetic control of protein binding using light-switchable nanobodies
a Size exclusion chromatography (SEC) for light-dependent protein separation. The column was wrapped with 450 nm blue LEDs to allow for direct illumination of the protein during the SEC run, or in aluminum foil to keep it in darkness. b, c SEC elution profile for LaM8-AK74 (in b) and LaM4 TK74 (in c) in light conditions; for additional runs in the light and dark see Supplementary Fig. 4a, b. Free mCherry, free OptoNB, and dark- and light-incubated OptoNB/mCherry mixtures are shown in the indicated curves. Shorter retention times indicate larger size and increased complex formation. d Schematic representing light-induced binding of mCherry to OptoNB-coated beads. His-tagged OptoNBs are immobilized on Ni-NTA agarose beads, while untagged mCherry is in the buffer solution surrounding the beads. A change in illumination conditions results in mCherry-OptoNB binding and a brighter bead surface in the mCherry channel. e, f Top panels: confocal mCherry images of beads coated with a mixture of His-tagged EGFP with His-tagged LaM8-AK74 in a 200:1 ratio (in e) or LaM8-GG15 in a 1000:1 ratio (in f). Beads were placed in 1 μM mCherry solution (in e) or 2 μM mCherry solution (in f). A 450 nm LED was toggled on and off (blue shading indicates LED illumination). Bottom panels: quantification of bead surface intensity during cycles of darkness and blue light illumination. Images are representative of three replicate experiments. g Representative bio-layer interferometry (BLI) traces for quantifying nanobody-protein binding and dissociation kinetics. Four phases indicate His-tagged OptoNB loading onto the Ni-NTA coated tips, equilibration in buffer, binding to different concentrations of soluble mCherry, and mCherry dissociation into buffer. h Raw data (solid lines) and best-fit traces simultaneously fit to simple mass-action kinetic binding model (dashed lines) are shown for the immobilized LaM8 nanobody binding to soluble mCherry at eight concentrations. Source data are available as a Source data file.
