Photoreactive bicyclic amino acids as substrates for mutant Escherichia coli phenylalanyl-tRNA synthetases
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Photoreactive bicyclic amino acids as substrates for mutant Escherichia coli phenylalanyl-tRNA synthetases. / Bentin, Thomas; Hamzavi, Ramin; Salomonsson, Johan; Roy, Hervé; Ibba, Michael; Nielsen, Peter E.
In: The Journal of Biological Chemistry, Vol. 279, No. 19, 07.05.2004, p. 19839-45.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Photoreactive bicyclic amino acids as substrates for mutant Escherichia coli phenylalanyl-tRNA synthetases
AU - Bentin, Thomas
AU - Hamzavi, Ramin
AU - Salomonsson, Johan
AU - Roy, Hervé
AU - Ibba, Michael
AU - Nielsen, Peter E.
PY - 2004/5/7
Y1 - 2004/5/7
N2 - Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested for in vitro charging of tRNA(Phe) and for protein mutagenesis via the phenylalanyl-tRNA synthetase variant alphaA294G that is able to facilitate in vivo protein synthesis using a range of para-substituted phenylalanine analogues. The results demonstrate that benzofuranylalanine, but not benzotriazolylalanine, is a substrate for phenylalanine tRNA synthetase alphaA294G, and matrix-assisted laser desorption ionization time-of-flight analysis showed it to be incorporated into a model protein with high efficiency. The in vivo incorporation into a target protein of a bicyclic phenylalanine analogue, as described here, demonstrates the applicability of phenylalanine tRNA synthetase variants in expanding the scope of protein engineering.
AB - Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested for in vitro charging of tRNA(Phe) and for protein mutagenesis via the phenylalanyl-tRNA synthetase variant alphaA294G that is able to facilitate in vivo protein synthesis using a range of para-substituted phenylalanine analogues. The results demonstrate that benzofuranylalanine, but not benzotriazolylalanine, is a substrate for phenylalanine tRNA synthetase alphaA294G, and matrix-assisted laser desorption ionization time-of-flight analysis showed it to be incorporated into a model protein with high efficiency. The in vivo incorporation into a target protein of a bicyclic phenylalanine analogue, as described here, demonstrates the applicability of phenylalanine tRNA synthetase variants in expanding the scope of protein engineering.
KW - Alanine/analogs & derivatives
KW - Amino Acids/chemistry
KW - Benzofurans/chemical synthesis
KW - Escherichia coli/enzymology
KW - Kinetics
KW - Mass Spectrometry
KW - Models, Chemical
KW - Mutagenesis
KW - Phenylalanine/chemistry
KW - Phenylalanine-tRNA Ligase/genetics
KW - Protein Binding
KW - Protein Engineering
KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
KW - Substrate Specificity
KW - Time Factors
KW - Triazoles/chemical synthesis
KW - Ultraviolet Rays
U2 - 10.1074/jbc.M401278200
DO - 10.1074/jbc.M401278200
M3 - Journal article
C2 - 15004015
VL - 279
SP - 19839
EP - 19845
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 19
ER -
ID: 203634099