Rational Design of Light-responsive Polypeptides, Using Non-standard Amino Acids

Photosensitive bioactive molecules have great potential in basic science and medical applications, since they facilitate an on-demand release of any encapsulated payload. Using light as a trigger for remote control allows spatio-temporal activation of biomolecules, without invasive procedures. Here we present rationally designed Elastin-like polypeptide (ELP), that phase-transition in response to light irradiation. ELPs are a family of stimulus-responsive protein polymers that comprises artificial repetitive polypeptides composed of a VPGXG repeat, where X can be any amino acid except proline. Therefore, the X-position is also permissive to non-standard amino acid (nsAA) incorporation. Notably, ELPs undergo a reversible soluble-to-insoluble phase transition at their lower critical transition temperature (Tt), which depends on the ELP composition. In this work, we aim to generate light-responsive ELPs by incorporation of azobenzene-containing non-standard amino acid (nsAA) in the X-position of the ELP sequence. Azobenzenes are a class of molecules known to undergo reversible light-based isomerization from trans to cis, which also impacts the polarity of the molecule. To incorporate multiple instances of azobenzene-nsAAs we utilized an improved orthogonal translation system (OTS) and genomically recoded organisms. We show that multi-site-specific incorporation of nsAAs into a polypeptide can facilitate the production of complex and precise biopolymers using a relatively simple methodology. Ultimately, the engineering of light-responsive phase transition in ELPs, using site-specific azobenzene incorporation, can be used as a basis for engineering an entire family of novel light-responsive biomaterials.