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.
Wang
Pin
P
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Tang
Yi
Y
Tirrell
David A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D006845:Hydrocarbons, Fluorinated / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D007376:Interleukin-2 / Q000737:chemistry / Q000378:metabolism*; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
D006845:Hydrocarbons, Fluorinated; D007376:Interleukin-2; D007532:Isoleucine; D000255:Adenosine Triphosphate; D013762:Tetrahydrofolate Dehydrogenase; D007533:Isoleucine-tRNA Ligase