Fig. 1: Engineering of light-controlled protein splicing.

a Design of photoswitchable intein based on insertion of a double VVD allosteric switch in gp41-1 intein. As a readout, mCherry split at position 156 was attached as N- and C-exteins (flanking protein fragments external to intein). Covalent binding of mCherry fragments results in proper folding and reconstitution of fluorescent mCherry. In darkness, the IntC is allosterically distorted (left). Illumination with 450 nm leads to homodimerization of VVD domains with subsequent restoration of the properly folded IntC (center). Intein-catalyzed protein splicing results in reconstitution of mCherry (right). b Schematics of the photoswitchable intein construct. c Positions of the double VVD insertion (yellow) shown on the structure of gp41-1 (purple) (6qaz). Linear schematic is shown below. d Screening of photoswitchable intein variants with the double VVD loop at different positions. HeLa cells were transfected with respective constructs, kept in the darkness or illuminated with 450 nm and analyzed by flow cytometry. mCherry fluorescence signals normalized to co-expressed mEGFP were analyzed. Respective light-to-dark contrasts are shown above the bars. Mean values for individual experiments and SD are shown (n = 3). e Testing of linkers 1 and 3 (shown in (b)) connecting the double VVD insert to the gp41-1 intein in HeLa cells. Cells were processed and analyzed as in (d). Mean values for individual experiments and SD are shown (n = 3). Arb. units, arbitrary units. Source data are provided as a Source Data file.