β‑Amino Acids Reduce Ternary Complex Stability and Alter the Translation Elongation Mechanism

Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to expand the chemical space available to biological therapeutics and materials, but existing technologies are still limiting. Addressing these limitations requires a deeper understanding of the mechanism of protein synthesis and how it is perturbed by nnAAs. Here we examine the impact of nnAAs on the formation and ribosome utilization of the central elongation substrate: the ternary complex of native, aminoacylated tRNA, thermally unstable elongation factor, and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (R)- and (S)-β² isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-β³-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (R)- and (S)-β²-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-β³-Phe stereoisomers were utilized inefficiently. (R)-β³-Phe but not (S)-β³-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include the consideration of the efficiency and stability of ternary complex formation.