TY - JOUR
T1 - Tuning the Properties of Protein-Based Polymers Using High-Performance Orthogonal Translation Systems for the Incorporation of Aromatic Non-Canonical Amino Acids
AU - Gueta, Osher
AU - Sheinenzon, Ortal
AU - Azulay, Rotem
AU - Shalit, Hadas
AU - Strugach, Daniela S.
AU - Hadar, Dagan
AU - Gelkop, Sigal
AU - Milo, Anat
AU - Amiram, Miriam
N1 - Funding Information:
This work was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation program, grant agreement 756996 and by the Israel Science Foundation (grant number 939\21) (to MA). MA gratefully acknowledges support from the Elaine S. and Alvin W. Wene Career Development Chair in Biotechnology Engineering. HS acknowledges support from the Emergency Postdoctoral Fellowships for Israeli Researchers in Israel.
Publisher Copyright:
Copyright © 2022 Gueta, Sheinenzon, Azulay, Shalit, Strugach, Hadar, Gelkop, Milo and Amiram.
PY - 2022/5/30
Y1 - 2022/5/30
N2 - 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.
AB - 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.
KW - elastin-like polypeptides (ELP)
KW - genetic code expansion
KW - non-canonical amino acids (ncAAs)
KW - resilin-like polypeptides (RLP)
KW - smart biomaterials
UR - http://www.scopus.com/inward/record.url?scp=85132423654&partnerID=8YFLogxK
U2 - 10.3389/fbioe.2022.913057
DO - 10.3389/fbioe.2022.913057
M3 - Article
C2 - 35711629
AN - SCOPUS:85132423654
SN - 2296-4185
VL - 10
JO - Frontiers in Bioengineering and Biotechnology
JF - Frontiers in Bioengineering and Biotechnology
M1 - 913057
ER -