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Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo

van Hest, Jan C. M. and Kiick, Kristi L. and Tirrell, David A. (2000) Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo. Journal of the American Chemical Society, 122 (7). pp. 1282-1288. ISSN 0002-7863. doi:10.1021/ja992749j.

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A set of eight methionine analogues was assayed for translational activity in Escherichia coli. Norvaline and norleucine, which are commercially available, were assayed along with 2-amino-5-hexenoic acid (2), 2-amino-5-hexynoic acid (3), cis-2-amino-4-hexenoic acid (4), trans-2-amino-4-hexenoic acid (5), 6,6,6-trifluoro-2-aminohexanoic acid (6), and 2-aminoheptanoic acid (7), each of which was prepared by alkylation of diethyl acetamidomalonate with the appropriate tosylate, followed by hydrolysis. The E. coli methionine auxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was used as the expression host, and translational activity was assayed by determination of the capacity of the analogue to support synthesis of the test protein dihydrofolate reductase (DHFR) in the absence of added methionine. The importance of amino acid side chain length was illustrated by the fact that neither norvaline (8) nor 7 showed translational activity, in contrast to norleucine (9), which does support protein synthesis under the assay conditions. The internal alkene functions of 4 and 5 prevented incorporation of these analogues into test protein, and the fluorinated analogue 6 yielded no evidence of translational activity. The terminally unsaturated compounds 2 and 3, however, proved to be excellent methionine surrogates:  ^1H NMR spectroscopy, amino acid analysis, and N-terminal sequencing indicated ∼85% substitution of methionine by 2, while 3 showed 90−100% replacement. Both analogues also function efficiently in the initiation step of protein synthesis, as shown by their near-quantitative occupancy of the N-terminal amino acid site in DHFR. Enzyme kinetics assays were conducted to determine the rate of activation of each of the methionine analogues by methionyl tRNA synthetase (MetRS); results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analogue incorporation in vivo is controlled by MetRS.

Item Type:Article
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URLURL TypeDescription
Kiick, Kristi L.0000-0001-8587-0301
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2000 American Chemical Society. Received August 2, 1999. Publication Date (Web): February 3, 2000. This work was supported by a grant from the Polymers and Genetics Programs of the U. S. National Science Foundation. The Netherlands Organization for Scientific Research (NWO) and DSM Research are acknowledged for unrestricted grants in support of our research. We are grateful to H. Jakubowski for plasmid pGG3. K.L.K thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship.
Funding AgencyGrant Number
Netherlands Organization for Scientific Research (NWO)UNSPECIFIED
National Defense Science and Engineering Graduate (NDSEG) FellowshipUNSPECIFIED
Issue or Number:7
Record Number:CaltechAUTHORS:HESjacs2000
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Official Citation:Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo Jan C. M. van Hest, Kristi L. Kiick, and David A. Tirrell Journal of the American Chemical Society 2000 122 (7), 1282-1288
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53379
Deposited By: Anne Hormann
Deposited On:06 Feb 2015 19:13
Last Modified:10 Nov 2021 20:01

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