Fig. 3: Design and optimization of PhoBIT2 as a light-ON switch.
From: Engineering of photo-inducible binary interaction tools for biomedical applications
a Schematic of PhoBIT2 design. CRY2 is fused to sspB to allow oligomerization, thereby enhancing its binding with the ssrA tag upon blue light stimulation. Mutations are introduced into both components to minimize the background interaction in the dark. The combination of ssrA2 (mutant A2C) and sspB2 (mutant A56F) is used to generate PhoBIT2, which displays the lowest background while retains strong light-inducible heterodimerization. b The 3D structure of the ssrA-sspB complex (PDB entry: 1TWB), with the optimized residues in PhoBIT2 (yellow, A2C in ssrA; magenta, A56F in sspB) indicated. c Schematic of a bioluminescence resonance energy transfer (BRET) assay design to screen ssrA mutants. ssrA mutants were anchored on mitochondria via fusion to Ntom20 with a Venus fluorescent tag, whereas mCherry-sspB-NanoLuc (NLuc) is located in the cytoplasm. The binary interaction between ssrA mutants and sspB allows the donor (NLuc) to transfer energy to the acceptor (Venus), resulting in a high BRET ratio. The Venus emission intensity over the summed emission intensity of NLuc emission is used to calculate the BRET ratio. d Quantification of BRET ratios for screened ssrA mutants. The BRET ratio for the wild-type ssrA-sspB interaction was normalized to 1 for comparison. e Heatmap showing the BRET ratio corresponding to each mutated position in the ssrA peptide following deep mutational scanning. f Left, cartoon illustration of a mitochondria translocation assay used to assess the relative strength of ssrA-sspB variant interaction in living cells. Right, heatmaps showing the fold-change of mitochondrial mCherry intensity for the indicated sspB variants following photostimulation. g Representative confocal images of HeLa cells co-expressing the indicated combinations of mitochondria-anchored ssrA mutants (Venus-tagged; green) and CRY2-fused sspB mutants (mCherry-tagged; red), before and after photostimulation. Scale bar, 10 μm. h Quantification of basal mitochondrial mCherry signal in the dark state (FDark; X-axis) and the fold-change of mitochondrial mCherry intensity upon light stimulation (FLight/Dark; Y-axis) during ssrA-sspB screening. CRY2olig-CIB1: HeLa cells co-expressing Ntom20-Venus-CIB1 and mCh-CRY2olig. (Source Data).
