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Incorporation of Trifluoroisoleucine into Proteins in Vivo

Wang, Pin and Tang, Yi and Tirrell, David A. (2003) Incorporation of Trifluoroisoleucine into Proteins in Vivo. Journal of the American Chemical Society, 125 (23). pp. 6900-6906. ISSN 0002-7863. doi:10.1021/ja0298287.

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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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Tirrell, David A.0000-0003-3175-4596
Additional Information:Copyright © 2003 American Chemical Society. Published In Issue June 11, 2003. Publication Date (Web): May 17, 2003. Received December 19, 2002. Acknowledgment. This work was supported by NIH grant R01-GM62523, and by the NSF Center for the Science and Engineering of Materials at Caltech. We are grateful to Lili Yang from David Baltimore’s laboratory at Caltech and Dr. Susan Kovats at City of Hope for assistance on the cell-proliferation assay. We thank Dr. Osamu Nureki at the University of Tokyo for providing coordinates of the crystal structure of IleRS complexed with isoleucine, and Kent Kirshenbaum for helpful discussions.
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NSF Center for the Science and Engineering of Materials at CaltechUNSPECIFIED
Issue or Number:23
Record Number:CaltechAUTHORS:20150116-102631096
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Official Citation: Incorporation of Trifluoroisoleucine into Proteins in Vivo Pin Wang,Yi Tang, and, and David A. Tirrell* Journal of the American Chemical Society 2003 125 (23), 6900-6906
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53819
Deposited By: Katherine Johnson
Deposited On:16 Jan 2015 18:38
Last Modified:10 Nov 2021 20:07

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