Tuning the Properties of Protein-Based Polymers Using High-Performance Orthogonal Translation Systems for the Incorporation of Aromatic Non-Canonical Amino Acids

Abstract

The incorporation of non-canonical amino acids (ncAAs) using engineered aminoacyl-tRNA synthetases (aaRSs) has emerged as a powerful methodology to expand the chemical repertoire of proteins. However, the low efficiencies of typical aaRS variants limit the incorporation of ncAAs to only one or a few sites within a protein chain, hindering the design of protein-based polymers (PBPs) in which multi-site ncAA incorporation can be used to impart new properties and functions. Here, we determined the substrate specificities of 11 recently developed high-performance aaRS variants and identified those that enable an efficient multi-site incorporation of 15 different aromatic ncAAs. We used these aaRS variants to produce libraries of two temperature-responsive PBPs—elastin- and resilin-like polypeptides (ELPs and RLPs, respectively)—that bear multiple instances of each ncAA. We show that incorporating such aromatic ncAAs into the protein structure of ELPs and RLPs can affect their temperature responsiveness, secondary structure, and self-assembly propensity, yielding new and diverse families of ELPs and RLPs, each from a single DNA template. Finally, using a molecular model, we demonstrate that the temperature-responsive behavior of RLPs is strongly affected by both the hydrophobicity and the size of the unnatural aromatic side-chain. The ability to efficiently incorporate multiple instances of diverse ncAAs alongside the 20 natural amino acids can help to elucidate the effect of ncAA incorporation on these and many other PBPs, with the aim of designing additional precise and chemically diverse polymers with new or improved properties.

Engineered bacterial host for genetic encoding of physiologically stable protein nitration

“…Figure 3 shows that high full length production yields can be achieved for ELP(5x ONB-Dopa). This is consistent with previous reports and also with quantification of small scale protein production yields (for the latter, see paragraph 3.5) ( Amiram et al, 2015 ; Gueta et al, 2022 ). Reporter fluorescence signals show that constructs with up to 60 stop codons can produce full length protein ( Figure 3B ).…”

“…The level of background suppression (stop codon readthrough with canonical amino acid incorporation) decreased by around 30% ( Figure 2B ). This behavior highlights that this amino acid substrate recognition is quite different compared to other Mj TyrRS mutants ( Dumas et al, 2015 ; Gueta et al, 2022 ). Reversion of Y32A generally resulted in complete inactivation of ncAA recognition or a drastic reduction in catalytic activity and fidelity for the corresponding ncAAs ( Antonczak et al, 2011 ).…”

“…Reporter fluorescence signals show that constructs with up to 60 stop codons can produce full length protein ( Figure 3B ). To date, at most 30 stop codons have been suppressed in vivo ( Amiram et al, 2015 ; Gueta et al, 2022 ) and 40 in vitro , respectively ( Martin et al, 2018 ). This is intriguing since it is well known that increasing the number of in-frame stop codons normally dramatically decreases the efficiency of translation.…”

Robust Photocontrol of Elastin-like Polypeptide Phase Transition with a Genetically Encoded Arylazopyrazole

“…We began by determining the ability of our previously evolved aaRSs, derived from the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS), to incorporate AAP (10 instances per protein) in the Escherichia coli strain C321.ΔRF1, which lacks all the native TAG codons and their associated release factor (RF-1). To this end, we utilized our previously described ELP-based reporter protein ELP­(10TAG)-GFP to evaluate the multisite incorporation of AAP in response to TAG codons (Table S1).…”

Orthogonal translation with 5‐cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding

“…However, the stringency of the negative selection cycle can lead to the removal of enzyme variants whose amino acid selectivity is sufficient in the presence of ncAA supplementation. In several instances, engineered aaRS variants were reported which allow the production of homogeneously ncAA‐labeled protein, despite leading to a background incorporation of canonical amino acids in absence of ncAA supply (Gueta et al, 2022). The utility of this subset of enzymes is obvious, but they would disappear from the aaRS pool during the negative selection cycle.…”