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

Abstract

AbstractSynthesis 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.