Genetically encoding light-responsive protein-polymers

Enabling researchers and clinicians to control and manipulate biological agents in space and time is a defining challenge in biology, bioengineering, and medicine. In this context, light is a unique external regulatory element because it can be precisely controlled in location, timing and amplitude. An ideal methodology for encoding and fine-tuning light-responsive behavior in proteins should permit the exact positioning of multiple, small, reversibly photo-switchable groups in the primary sequence of the protein.

Of particular interest to us are two families of unstructured proteins-based biomaterials (PBBs), Elastin and Resilin Like Polypeptides (ELPs and RLPs), which are stimuli responsive, biocompatible biomolecules, that undergo a reversible soluble to insoluble, phase transition in a lower and upper critical transition temperature (LCST/UCST) respectively. Our hypothesis is that by incorporating photoswitchable unnatural amino acids (uAAs) in these PBB sequences, in a site-specific manner, we will also be able to genetically encode a light-based phase transition, enabling us to create polymer based photoswitches.

To this end, an orthogonal aminoacyl tRNA synthetase capable of multi-site incorporation of UV- or visible-light-responsive azobenzene-bearing uAAs in a single protein-polymer was evolved using a directed evolution approach. This translation system enabled efficient and accurate incorporation of up to 12 instances of the various light-responsive uAAs in ELPs and RLPs. Azobenzene-containing ELPs and RLPs were shown to be capable of isothermal, reversible, light-mediated soluble-to-insoluble phase transition, with up to a 12 °C difference in the transition temperature upon cis-to-trans azobenzene isomerization. Furthermore, the incorporation of azobenzene-uAAs in ELP diblock-copolymers enabled the creation of light-responsive self-assembled nanostructures. We envision that Proteins and PBBs with such tailored and tunable light-responsive behaviors could then be manipulated by light in both space and time to sequester or release biomolecules on demand, elucidate complex cellular behaviors, and to use as scaffolds for sophisticated extra- and intra-cellular factories.

Israeli, B., Strugach, D. S. et al. (2021). Genetically Encoding Light-Responsive Protein-Polymers Using Translation Machinery for the Multi-Site Incorporation of Photo-Switchable Unnatural Amino Acids. Advanced Functional Materials, 2011276