Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages

Abstract

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an α-amino acid or α-hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non-α-amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse α-hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an α-hydroxy acid monomer into a protein translatedin vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an α-peptide backbone directly into a β-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone β-amino acid linkage has been introduced site-selectively into a protein in a cell.

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

“…Overall, the combined biochemical and computational approach reported here provides the first example of orthogonal cellular translation through a β-backbone and an important steppingstone towards cell-based synthesis of β 2 -HA/α-AA hybrids with new-to-nature functionalities. They also improve our understanding of how the WT E. coli ribosome accommodates substrates with non-native stereochemistry and backbone configurations and suggest that translation factor 47 and/or ribosomal engineering, 20,48,49 as well as alternative approaches, 50 may be needed to achieve robust levels of β-linkages within proteins produced in cells.…”

Incorporation of Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase

“…Such next-generation molecules provide strategies for improved biologic therapies, tools for bioremediation, and plastic-like materials that biodegrade. But progress toward sequence-defined non-protein materials l has been exceptionally slow. − Although most elements of the translational machinery tolerate even wildly divergent α-amino acid side chains, altered backbones are tolerated predominantly in vitro , at small scale, under nonphysiological conditions, and with efficiencies and fidelities that have not been rigorously evaluated.…”

“…They also deepen our understanding of how the WT E. coli ribosome accommodates substrates with non-native stereochemistry and backbone configurations (or not) and suggest that translation factor , and/or ribosome engineering, ,, as well as alternative chemical and biochemical approaches, , may be needed to achieve robust levels of expanded backbone linkages within proteins produced in cells.…”

“…Despite enormous interest in sequence-defined biomaterials containing non-α-amino acid monomers, progress towards their in vivo biosynthesis has been exceptionally slow. − There exists one report in which a single β 2 -ester has been introduced into a protein using the wild-type E. coli ribosome in vitro , but there are no examples in which any β 2 -hydroxy acid has been introduced into a ribosomal product in vivo .…”

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

“…More than 200 different non-natural α-amino acids (nnAA) and a handful of α-hydroxy acids can be incorporated into proteins [1][2][3][4][5] . There are now several examples of β 2and/or β 3 -monomers that have been introduced into proteins in cells, either directly [6][7][8] or via rearrangement 9 . Robust methods of α-and non-α nnAA incorporation into proteins is expected to promote the development of new tools to probe structure-function relationships, discover catalysts, and advance therapeutic approaches.…”