Fig. 1: Design and construction of the GPlad system.
From: De novo designed protein guiding targeted protein degradation
a Schematic representation of GPlad. First, a guide protein (GP) is fused with a marking protein (MP) via a linker, forming a GP-MP complex. GP binds the target protein and brings MP close to the target protein. The MP labels the target protein, and then the labeled target protein is subsequently recognized and degraded by the corresponding proteases. b Left: Western blot (WB) analysis showing McsBE121A and McsB-mediated arginine phosphorylation of mKate2 guided by 1/1′ heterodimer. Right: WB analysis showing PafAH123A and PafA-mediated ubiquitin-like modification of mKate2 guided by 1/1′ heterodimer. c Flow cytometry analysis of intracellular mKate2 fluorescence intensity at 0, 3, and 6 h after induction of McsB or PafA expression. P-value was determined by a two-tailed unpaired Student’s t-test. ****p < 0.0001. d WB analysis of intracellular mKate2 abundance after 6 h of induction using the McsB or PafA system. e Visualization of the selected binding interface of the GP on the mKate2 surface. Red indicates hydrophobic amino acids (Ala, Val, Thr, Ile, Pro, Leu, Met, Phe, Tyr, Trp) on the mKate2 surface, yellow represents PatchDock residues. f Left: Design model of GPmKate2 in complex with mKate2. Right: Confocal imaging of the co-location of GPmKate2-McsB-eGFP (Green) with mKate2-PopZ (Red) at cell poles. g Plasmid map of the GPlad degradation system, with GP-McsB placed under the Ptet promoter and ClpCP under the PLtetO-1 promoter. h GP-guided arginine phosphorylation of McsBE121A and McsB for mKate2. i Fluorescence changes at 0, 3, and 6 h after GPlad induction, assessed by flow cytometry. j Intracellular mKate2 changes at 0, 3, and 6 h after GPlad induction, using laser scanning confocal microscopy. k Intracellular mKate2 abundance at 0 and 6 h after GPlad induction, detected by WB. All microscopy experiments were performed in triplicate with similar results. All experiments are presented as mean ± s.d. from three biologically independent replicates. Source data are provided as a Source Data file. (a) is created in BioRender https://BioRender.com/3ihz25i.
