Incorporation of multiple β²-backbones into a proteinin vivousing an orthogonal aminoacyl-tRNA synthetase

Abstract

Synthesis of sequence-defined biomaterials whose monomer backbones diverge from canonical α-amino acids represents the next frontier in protein and biomaterial evolution with the potential to yield better biological therapeutics, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials are β-hydroxy acids. Many natural products contain isolated β-esters, and polymeric β-esters are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that β2-hydroxy acids possessing both(R)and(S)absolute configuration are excellent substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymesin vitro, and that(S)-β2-hydroxy acids are substratesin cellulo. Using theMaPylRS/MatRNAPylpair, in conjunction with wild-typeE. coliribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two(S)-β2-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed enantioselective preference of the ribosome for the(S)-β2-hydroxy acid backbone and mechanistic insights that inform future ribosomal engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that accepts a β-backbone substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers producedin cellulo.

β-amino acids reduce ternary complex stability and alter the translation elongation mechanism

“…However, their reaction yields are seldom reported and only a minor fraction of published studies focus on relative rates or mechanistic bottlenecks 16 . In cellulo studies that show incorporation of nnAAs outside of α-amino or α-hydroxy acid are far fewer [6][7][8]76 . Parallel in vitro and in cellulo investigations are likely to be most informative.…”

“…β 3 -( p -Br)-Phe has also been incorporated into DHFR in E. coli cells expressing a ribosome containing a remodeled PTC 6 . A β 2 -hydroxy analogue of N ε -Boc-L-α-Lysine has also been incorporated into sfGFP by a wild-type E. coli strain 7 , although the yield of protein was far lower based than expected from the in vitro activity of the aaRS. Kinetic simulations of ternary complex formation reveal that this discrepancy is likely explained by the presence of elevated EF-Tu and tRNA concentrations in the cell that help drive ternary complex formation despite significant reductions in stability ( Fig.…”

“…Moreover, one β 3 -Phe derivative 6 , three β 3 -aryl derivatives 8 , and one β 2 -hydroxy acid 7 have been introduced into proteins in cells. Yet in none of these cases was the level of incorporation especially high, perhaps because of impaired delivery to the ribosome by EF-Tu 52,53 .…”

“…Such strides have permitted the incorporation of α-amino acids with distinct non-natural side chains into otherwise native proteins 11,14 . However, the translational machinery did not evolve to support the translation of components with alternative backbones, and significant evidence exists that multiple kinetic bottlenecks likely exist 7,15,16 . Here we identify kinetic bottlenecks that exist during the delivery and accommodation of acylated tRNA by the ribosome.…”

A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases

“…The key bottleneck limiting the progress on this front is the lack of engineered aaRSs capable of efficiently charging such monomers. In the handful of examples, where such exotic monomers were incorporated into proteins in E. coli , such as β 3 - p -Bromo-homophenylalanine 26 and a β 2 -hydroxy acid, 29 tRNA acylation relied on promiscuous recognition that certain wild-type aaRSs fortuitously exhibited for these substrates. The ability to systematically engineer aaRSs to charge a wider variety of noncanonical monomers with high fidelity and efficiency is needed to overcome this limitation.…”