Genetically Encoding Light-Responsive Protein-Polymers Using Translation Machinery for the Multi-Site Incorporation of Photo-Switchable Unnatural Amino Acids

A general and versatile technology to engineer light-responsive protein-based biomaterials can enable the manipulation and interrogation of proteins, pathways, and cells, and it will assist the design of "smart" light-responsive biomaterials. This study reports the evolution of chromosomal aminoacyl-tRNA synthetases (aaRSs) for azobenzene-bearing unnatural amino acids (uAAs) with up to approximate to 40-fold increased protein production and improved fidelity, as compared with a previously described aaRS. The evolved translation systems enable efficient and accurate incorporation of up to 10 instances of the various light-responsive uAAs in elastin-like polypeptides (ELPs). Azobenzene-containing ELPs are capable of isothermal, reversible, light-mediated soluble-to-insoluble phase transition, with up to a 12 degrees C difference in the ELP transition temperature upon cis-to-trans azobenzene isomerization. Furthermore, the incorporation of azobenzene-uAAs in ELP diblock-copolymers enables the creation of light-responsive self-assembled nanostructures. Finally, light-responsive resilin-inspired polymers are also generated by multi-site azobenzene-incorporation. The translation machinery evolved in this study can be used for the multi-site incorporation of azobenzene moieties at the polypeptide level and constitute a universal methodology for the design of light-responsive proteins and additional families of protein-based biomaterials with customized and tunable light-responsive behavior.