Stereoselective Incorporation of an Unsaturated Isoleucine Analogue into a Protein Expressed in E. coli

Abstract

The unsaturated amino acid 2‐amino‐3‐methyl‐4‐pentenoic acid (E‐Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS‐E‐Ile and SR‐E‐Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS‐E‐Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR‐E‐Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS‐E‐Ile was activated by the isoleucyl‐tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR‐E‐Ile was activated approximately 100‐times more slowly than SS‐E‐Ile.

Atomistic and continuum modeling of mechanical properties of collagen: Elasticity, fracture, and self-assembly

“…We use the crystal structure PDB ID 1QSU, with 1.75 Å resolution, as reported by Kramer and coworkers. 13 The 1QSU is a triplehelical collagen-like molecule with sequence (Pro-HypGly) 4 -Glu-Lys-Gly-(Pro-Hyp-Gly) 5 .…”

“…[1][2][3][4][5][6] Materials based on proteins hold particular promise because of their great flexibility in usage and their applications. Genetically engineered biopolymers based on recombinant DNA technologies have been developed by Tirrell and coworkers [1][2][3][4][5] for various applications, including pH sensitive hydrogels. 2 This is an example for the vanishing borderline between technology and biology, enabling new critical breakthroughs for novel material concepts, allowing translation of nature's structural concepts into engineered materials.…”

“…2 This is an example for the vanishing borderline between technology and biology, enabling new critical breakthroughs for novel material concepts, allowing translation of nature's structural concepts into engineered materials. [1][2][3][4][5][6] Together with such new experimental techniques for synthesis and characterization, [1][2][3][4][5][6] a sound theoretical understanding and the availability of accurate models are essential for developing engineering applications of such complex materials. The properties of biological materials are determined from complex interaction of individual scale-specific properties and their interactions.…”

Posttranslational Modification of Proteins Incorporating Nonnatural Amino Acids

“…By using RSI, selenomethionine [120], difluoromethionine [125], trifluoromethionine [126], norleucine [123,127], homopropargylglycine (Hpg) [128], azidohomoalanine (Aha) [129], homoallylglycine (Hag) [130], and methoxinine [127] have been incorporated using methionyl-tRNA synthetase (MetRS); per-thiaproline, 3 [131,132]; trifluorovaline (TfV), 2-amino-3-methyl-4-pentenoic acid have been incorporated using isoleucyl-tRNA synthetase (IleRS) [133][134][135]; o-fluorophenylalanine, m-fluorophenylalanine, pFF [136], 2-pyridylalanine, 3-pyridylalanine, and 4-pyridylalanine [137] have been incorporated using PheRS; 4-aminotryptophan (4AW), 5-aminotryptophan (5AW), 4-fluorotryptophan (4FW), 6-fluorotryptophan (6FW), 5-hydroxytryptophan (5HW), 7AzW have been incorporated using TyrRS [138][139][140][141]; and trifluoroleucine has been incorporated using leucyl-tRNA synthetase (LeuRS) [142,143] in E. coli (Table 12.3). Budisa and coworkers have explored RSI for the integration of Hpg and norleucine into the superoxide dismutase (SOD) protein in yeast [123].…”

Semisynthetic Enzymes

“…The yields of purified proteins expressed in this method vary widely from 10 to 100 mg/l of culture depending on the amino acid analog used as well as the recombinant protein and the purification method. Although this simple method has succeeded in reassigning the sense codons of different amino acids such as Trp [28,29], Met [30,31], Leu [32], Ile [33,34], Phe [35], Pro [36], and His [37] in recombinant proteins to many amino acid analogs, it has failed to incorporate other noncanonical amino acids in many cases. The fidelity of protein synthesis in the cell is controlled in large part by the aaRS [26], either by activation of the amino acid or by ligation of the activated amino acid to its cognate tRNA.…”