Incorporation of Multiple β 2 -Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase.

The programmed synthesis of sequence-defined biomaterials whose monomer backbones diverge from those of canonical α-amino acids represents the next frontier in protein and biomaterial evolution. Such next-generation molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials includes β-hydroxy acids. Many natural products contain isolated β-hydroxy acid monomers, and polymers of β-hydroxy acids (β-esters) are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that β ² -hydroxy acids possessing both ( R ) and ( S ) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that ( S )-β ² -hydroxy acids are substrates in cellulo . Using the orthogonal Ma PylRS/ Ma tRNA ^Pyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two ( S )-β ² -hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the ( S )-β ² -hydroxy acid and provide mechanistic insights that inform future engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that acylates tRNA with a β ² -hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo .
Competing Interests: The authors declare the following competing financial interest(s): N.X.H. and A.S. are coinventors on an international patent application that incorporates methods outlined in this manuscript.
(© 2024 The Authors. Published by American Chemical Society.)