Incorporation of trifluoroisoleucine into proteins in vivo.
Two fluorinated derivatives of isoleucine: d,l-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and d,l-2-amino-5,5,5-trifluoro-3-methyl pentanoic acid (5TFI, 3) were prepared. 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine. Incorporation of 5TFI was confirmed by tryptic peptide analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product. Amino acid analysis showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI. Measurement of the rate of activation of 5TFI by the E. coli isoleucyl-tRNA synthetase (IleRS) yielded a specificity constant (k(cat)/K(m)) 134-fold lower than that for isoleucine. 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions. The concentration of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC(50) = 2.70 ng/mL). The maximal responses are equivalent for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures. 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro.
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CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N CHEMICAL GENE-Y GENE-Y CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL
5TFI d,l-2-amino-3-trifluoromethyl pentanoic acid Amino acid isoleucine 5TFI 5TFI isoleucyl isoleucine 5TFI 3TFI d,l-2-amino-5,5,5-trifluoro-3-methyl pentanoic acid isoleucine trifluoroisoleucine mIL-2 cytokines IleRS murine dihydrofolate reductase mDHFR E. coli isoleucyl-tRNA synthetase IleRS cytokine 3TFI murine interleukin-2 mIL-2 5TFI 5TFI dihydrofolate isoleucine isoleucine 5TFI isoleucine