F. Aaron Cruz-Navarrete, Wezley C. Griffin, Yuk-Cheung Chan, Maxwell I. Martin, Jose L. Alejo, Ryan A. Brady, S. Kundhavai Natchiar, Isaac J. Knudson, Roger B. Altman, Alanna Schepartz, Scott J. Miller* and Scott C. Blanchard*,
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By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (<i>R</i>)- and (<i>S</i>)-β<sup>2</sup> isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (<i>R</i>)- and (<i>S</i>)-β<sup>3</sup>-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (<i>R</i>)- and (<i>S</i>)-β<sup>2</sup>-Phe-charged tRNAs were not utilized by the ribosome, while (<i>R</i>)- and (<i>S</i>)-β<sup>3</sup>-Phe stereoisomers were utilized inefficiently. (<i>R</i>)-β<sup>3</sup>-Phe but not (<i>S</i>)-β<sup>3</sup>-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.</p><p >β-Phe amino acids perturb several critical steps of the protein synthesis mechanism, revealing previously unappreciated bottlenecks that limit noncanonical amino acid incorporation into proteins.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7000,"publicationDate":"2024-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00314","citationCount":"0","resultStr":"{\"title\":\"β-Amino Acids Reduce Ternary Complex Stability and Alter the Translation Elongation Mechanism\",\"authors\":\"F. Aaron Cruz-Navarrete, Wezley C. Griffin, Yuk-Cheung Chan, Maxwell I. 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By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (<i>R</i>)- and (<i>S</i>)-β<sup>2</sup> isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (<i>R</i>)- and (<i>S</i>)-β<sup>3</sup>-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (<i>R</i>)- and (<i>S</i>)-β<sup>2</sup>-Phe-charged tRNAs were not utilized by the ribosome, while (<i>R</i>)- and (<i>S</i>)-β<sup>3</sup>-Phe stereoisomers were utilized inefficiently. (<i>R</i>)-β<sup>3</sup>-Phe but not (<i>S</i>)-β<sup>3</sup>-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. 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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.
β-Phe amino acids perturb several critical steps of the protein synthesis mechanism, revealing previously unappreciated bottlenecks that limit noncanonical amino acid incorporation into proteins.
期刊介绍:
ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.