Fig. 1. Initial screen for light-controllable opto-nanobodies (OptoNBs).

a Schematic of approach. By insertion into a solvent-exposed turn or loop, the light-switchable AsLOV2 domain (blue) could modulate the conformation of a nanobody (gray), thus allosterically altering its ability to bind to a target protein (red). Cytosolic iRFP-fused OptoNBs were assayed for translocation to membrane-bound mCherry in the presence or absence of blue light. b Positions targeted for LOV domain insertions mapped onto the crystal structure of an anti-GFP minimizer nanobody (PDB ID: 3G9A). Spheres indicate the residues between which the LOV domain was inserted. Loops of interest and the hypervariable complementarity-determining regions (CDRs) are colored according to the legend. c Representative images for all LOV insertions. HEK293 cells expressing membrane-tethered mCherry (mCherry-CAAX) and cytosolic OptoNB-iRFP (OptoNB) are shown. d Quantification of light-induced change in cytosolic intensity for each OptoNB variant in (c). An increase in cytosolic OptoNB fluorescence corresponds to light-induced dissociation from membrane-bound mCherry, and vice versa for light-induced decrease in cytosolic iRFP. Error bars indicate mean ± SEM for n = 8 cells per variant. e Images before (gray box) and after (blue box) light stimulation for HEK293T cells expressing mCherry-CAAX and either of two OptoNB variants, LaM8-AK74 and LaM8-GG15, showing light-dependent changes in OptoNB localization. Images are representative of three replicate experiments. Scale bars: 10 μm. Source data are available as a Source data file.