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 vastly expand the chemical space available to biological therapeutics and materials. Existing technologies limit the identity and number of nnAAs than can be incorporated into a given protein. Addressing these bottlenecks requires deeper understanding of the mechanism of messenger RNA (mRNA) templated protein synthesis and how this mechanism is perturbed by nnAAs. Here we examine the impact of both monomer backbone and side chain on formation and ribosome-utilization of the central protein synthesis substate: the ternary complex of native, aminoacylated transfer RNA (aa-tRNA), thermally unstable elongation factor (EF-Tu), and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements, we reveal the dramatic effect of monomer backbone on ternary complex formation and protein synthesis. Both the (R) and (S)-β² isomers of Phe disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-β³-Phe reduce ternary complex stability by approximately one 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. The reduced affinities of both species for EF-Tu ostensibly bypassed the proofreading stage of mRNA decoding. (R)-β³-Phe but not (S)-β³-Phe also exhibited order of magnitude defects in the rate of substrate translocation after mRNA decoding, in line with defects in peptide bond formation that have been observed for D-α-Phe. 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 consideration of the efficiency and stability of ternary complex formation.