Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo

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.

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: Structural and mechanistic implications of p17 myristoylation

“…Several techniques for site-specific unnatural modifications of proteins are currently in practice, including total chemical synthesis (32,38,39), in vitro translation (40 -42), auxotrophic bacterial expression (43)(44)(45)(46), enzymatic semisynthesis (47)(48)(49), native chemical ligation (30,50), expressed protein ligation (51), and, more recently, expression in Escherichia coli by using novel orthogonal tRNA͞synthetase pairs (52,53). Among these methods, total chemical synthesis coupled with native chemical ligation has proven to be a robust and versatile technique for the production of small-to medium-sized proteins (54).…”

Protein-based materials, toward a new level of structural control

“…Homoallylglycine and homopropargylglycine are especially effective as methionine surrogates. 68,71 Although trans-crotylglycine is recognized by the methionyl-tRNA synthetase, increasing the synthetase activity of the host is required for protein production. 72,73 The possibility of using mutant synthetases has also been demonstrated for p-bromophenylalanine 74 and azatyrosine.…”

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

“…Tirrell, Fournier and colleagues became interested in the use of unnatural amino acids to produce homogeneous, biopolymeric materials (periodic proteins) [118]. For example, 3-thienylalanine, azidohomoalanine, trifluoroisoleucine, hexafluoroleucine, homoallylglycine, and homopropargylglycine have been inserted into such proteins [119][120][121][122][123].…”