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Cited by 267

The DOI matches its Crossref record; 57 of the 57 references it cites were found in a registry or an index.

Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo

van Hest, Jan C. M., Kiick, Kristi L., Tirrell, David A.·2000·Journal of the American Chemical Society
DOI 10.1021/ja992749j
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Abstract

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: 1 H 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.

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Metadata

Authors
van Hest, J. C. M., Kiick, K. L., & Tirrell, D. A.
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2000-02-01
Volume
122
Issue
7
Pages
1282-1288
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/ja992749j
OpenAlex
W1982205639
Cited by
267
References
57
FWCI
2.59
Type
journal article
Language
en
Open access
closed

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