Fig. 1: DARPins used in Ubox-based bioPROTACs and degradation rate determination by fluorescence measurement.
From: Molecular features defining the efficiency of bioPROTACs
a Sequence alignment of eGFP-binding DARPins. The library consensus is shown in bold letters on the top. Randomized positions are highlighted in gray and labeled with character “X”. Mutations outside of randomized regions, acquired by directed evolution, are colored in yellow. A deletion in the 1st repeat of DP9 is colored in red. b Dendrogram of DARPin sequences created in CLC with tree construction method of neighbor joining and Jukes-Cantor protein distance measure. c Model of a CHIP-based dimeric bioPROTAC/eGFP complex. The monomer bioPROTAC structures were produced using AlphaFold and consist of the N-terminal DARPin (blue), followed by the CHIP E3 ligase coiled-coil region (light and dark gray) and Ubox-domain (light and dark yellow) that interact with E2 enzymes (not shown). The orientation of the two monomeric CHIPΔTPR domains is taken from the structure PDB ID: 2F42. The DARPin:GFP orientation is derived from PDB ID: 5MAD. d–f Representative single cell fluorescence signals of cells injected with specific analyte (respective left graphs) and mean rate constant derived from exponential decay fit of the single cell fluorescence signals (respective right graphs); error bars represent standard deviations. d GS-eGFP injection from one exemplary experiment. e DP6-bioPROTAC+GS-eGFP. f DP6-bioPROTAC+GS-eGFP with added proteasome inhibitor.
