Binding, Release, and Detection of Ligands by Ultrabithorax Protein‐Based Materials

Abstract

Proteins can bind, release, and detect molecules with unparalleled specificity. Incorporating proteins into materials can confer the ability to recognize specific ligands. We have leveraged the ability of materials composed of the Drosophila transcription factor Ultrabithorax (Ubx), a partially intrinsically disordered protein, to bind ligands, with the goals of delivering DNA to cells and creating biosensors. DNA binding and delivery is accomplished via the Ubx homeodomain, which retains its ability to recognize specific DNA sequences in Ubx materials. Ubx materials reversibly bind DNAs harboring the Ubx target DNA sequence and can protect these DNAs from degradation. DNA sequences with multiple binding sites have a slower rate of release from Ubx fibers. Ubx fibers can deliver DNA to transform E. coli, sustaining DNA release for up to 60 days.To build a biosensor, an additional protein (LOO8GFP) is incorporated into the materials via gene fusion. In this approach, the loo8gfp gene is placed adjacent to the ubx gene without intervening stop codons. When transformed into bacteria, this fused gene produces a LOO8GFP‐Ubx fusion protein. The intrinsically disordered regions of Ubx enable LOO8GFP incorporation without disrupting materials assembly or structure. The proof‐of‐concept sensor system is leave‐one‐out GFP (LOO8GFP), in which the 8th β‐strand has been ‘left out’ of circularly permuted GFP. Without this β‐strand the GFP is unable to fold properly and is not fluorescent. Fluorescence can be reconstituted with the addition of the left‐out peptide, making this construct a self‐reporting biosensor for the missing piece of its sequence. However, LOO8GFP can oligomerize, which (i) hampers ligand binding and (ii) leads to fluorescence in the unbound state. Ubx offers a transparent support system for LOO8GFP that maintains LOO8GFP stability and binding affinity while preventing LOO8GFP aggregation. To assess the utility of this composite material, we are examining assembly of LOO8GFP‐Ubx fibers, measuring binding affinities, and quantifying on‐ and off‐rates for the left‐out peptides. In the long term, the LOO8GFP‐Ubx system can be engineered to bind and detect other peptide sequences. Thus, Ubx materials can be engineered for variety of potential functions and uses based on incorporating bioactive DNAs and/or proteins.