Genetically Encoded 3,4-Ethylenedioxythiophene (EDOT) Functionality for Fabrication of Protein-Based Conductive Polymers

Genetic code expansion provides powerful strategies to improve the properties of protein-based materials. One novel application of this technique is to genetically incorporate an electroactive functional group into proteins, which can be subsequently polymerized into conductive polymers, enabling fabrication of various protein–conductive polymer hybrids that are widely applicable in bioelectronics. To this end, we developed a technique to incorporate an amino acid bearing 3,4-ethylenedioxythiophene (EDOT), the monomer precursor of a well-known conductive polymer PEDOT. In Chapter 1, we review the basics of protein-based materials and genetic code expansion technology. We also highlight some examples where genetic code expansion was used for the development of protein-based materials. Finally, we discuss applications of protein and peptide-based materials in bioelectronics. In Chapter 2, we describe our effort to incorporate an amino acid bearing EDOT group (EDOT-Ala) designed as an analogue of aromatic canonical amino acids. We synthesized EDOT-Ala in three steps of organic reaction, and evaluated the activity of known aminoacyl-tRNA synthetase (aaRS) variants for EDOT-Ala. In addition, we performed evolution of aaRS for EDOT-Ala using two different evolution techniques: cell viability- based approach and phage-assisted approach. Although the evolution experiment did not yield an aaRS variant that can incorporate EDOT-Ala, the results presented in this chapter provide valuable information for engineering of aaRS and incorporation of non-canonical amino acids (ncAA) with bulky functional groups. In Chapter 3, we describe the incorporation of another EDOT-functionalized amino acid (EDOT-Lys) designed as an analogue of a canonical amino acid pyrrolysine (Pyl). When we co-expressed a GFP reporter and a mutant pyrrolysyl-tRNA synthetase (PylRS) in E. coli in the presence of EDOT-Lys, the cells exhibited strong fluorescence as an indication of successful incorporation of EDOT-Lys into GFP. We further confirmed the incorporation using MALDI-TOF mass spectrometry. In Chapter 4, we describe the electropolymerization of a model protein XTEN that carries genetically incorporated EDOT-Lys (XTEN-E49am). We performed electropolymerization of XTEN-E49am in the presence of a self-doping EDOT monomer (EDOT-S) by cyclic voltammetry. The solution formed dark blue solids immediately after the potential cycles. The composition of the product was determined by FT-IR spectroscopy, suggesting that one protein is found per 12.5 monomer units of PEDOT. In addition, we investigated the effect of amino acids located adjacent to EDOT-Lys. Although the presence of cysteine (Cys), lysine (Lys), methionine (Met), arginine (Arg), and tryptophan (Trp) located adjacent to EDOT-Lys had an impact on the electropolymerization of model peptides, XTEN proteins carrying these adjacent residues (XTEN-E49am-G50Z; Z = Cys, Lys, Met, Arg, Trp) were electropolymerized with EDOT-S without noticeable effect from these adjacent residues, indicating that the EDOT-Lys residues in proteins undergo electropolymerization with EDOT-S in different chemical environments. In Chapter 5, we describe the oxidative chemical polymerization of XTEN proteins and model peptides. When XTEN-E49am was polymerized with EDOT-S by addition of ammonium persulfate (APS) and iron(III) chloride (FeCl3), the solution yielded dark blue solids. To evaluate the reactivity of the EDOT-Lys residue in the protein, we reacted the protein with an end-capped EDOT derivative (EDOT-cap). MALDI-TOF mass spectrometry revealed the appearance of new peaks corresponding to the addition of one or two EDOT-caps to the protein, suggesting that EDOT-Lys residue in the protein can react with EDOT derivatives. We also investigated the effect of adjacent amino acids using a series of model peptides. In polymerization using APS without FeCl3 catalyst, peptides carrying basic amino acids (His, Lys, Arg) adjacent to EDOT-Lys showed enhanced polymerization compared to the ones carrying neutral and acidic adjacent residues. All the tested peptides polymerized well when FeCl3 was added as a catalyst. The results presented in this chapter provide valuable insights into synthesis of protein–PEDOT conjugates via oxidative chemical polymerization.