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Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor.
Biochemistry. 2016 Jan 26; 55(3):618-28.B

Abstract

The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA(Tyr) cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA(Tyr) used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA(Tyr) suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA(Tyr) amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA(Tyr) used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid Km values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA(Tyr) may enhance future incorporation of many ncAAs by engineered TyrRS enzymes.

Authors+Show Affiliations

Department of Chemistry, Portland State University , P.O. Box 751, Portland, Oregon 97207, United States. Department of Biochemistry & Molecular Biology, Oregon Health & Sciences University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.Department of Biochemistry and Biophysics, Oregon State University , 2011 Agriculture and Life Sciences Building, Corvallis, Oregon 97331, United States.Department of Biochemistry and Biophysics, Oregon State University , 2011 Agriculture and Life Sciences Building, Corvallis, Oregon 97331, United States.Department of Chemistry, Portland State University , P.O. Box 751, Portland, Oregon 97207, United States. Department of Biochemistry & Molecular Biology, Oregon Health & Sciences University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

26694948

Citation

Rauch, Benjamin J., et al. "Improved Incorporation of Noncanonical Amino Acids By an Engineered tRNA(Tyr) Suppressor." Biochemistry, vol. 55, no. 3, 2016, pp. 618-28.
Rauch BJ, Porter JJ, Mehl RA, et al. Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor. Biochemistry. 2016;55(3):618-28.
Rauch, B. J., Porter, J. J., Mehl, R. A., & Perona, J. J. (2016). Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor. Biochemistry, 55(3), 618-28. https://doi.org/10.1021/acs.biochem.5b01185
Rauch BJ, et al. Improved Incorporation of Noncanonical Amino Acids By an Engineered tRNA(Tyr) Suppressor. Biochemistry. 2016 Jan 26;55(3):618-28. PubMed PMID: 26694948.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor. AU - Rauch,Benjamin J, AU - Porter,Joseph J, AU - Mehl,Ryan A, AU - Perona,John J, Y1 - 2016/01/08/ PY - 2015/12/24/entrez PY - 2015/12/24/pubmed PY - 2016/6/15/medline SP - 618 EP - 28 JF - Biochemistry JO - Biochemistry VL - 55 IS - 3 N2 - The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA(Tyr) cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA(Tyr) used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA(Tyr) suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA(Tyr) amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA(Tyr) used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid Km values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA(Tyr) may enhance future incorporation of many ncAAs by engineered TyrRS enzymes. SN - 1520-4995 UR - https://www.unboundmedicine.com/medline/citation/26694948/Improved_Incorporation_of_Noncanonical_Amino_Acids_by_an_Engineered_tRNA_Tyr__Suppressor_ L2 - https://dx.doi.org/10.1021/acs.biochem.5b01185 DB - PRIME DP - Unbound Medicine ER -