Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in
            <i>Escherichia coli</i>

Chatterjee, Abhishek; Xiao, Han; Schultz, Peter G.

doi:10.1073/pnas.1212454109

Cited by 102 publications

(98 citation statements)

References 33 publications

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“…We have altered the identity of a sense codon of an organism. This alteration is a significant shift from previous studies, where stop codons were partially reassigned to both canonical and artificial amino acids (15,18). Our data show that the genetic code is evolvable and that codon ambiguity provides the mechanism for such evolvability.…”

Section: Discussionmentioning

confidence: 34%

“…The flexibility of the genetic code is further highlighted by the in vivo incorporation of artificial amino acids into recombinant proteins of Escherichia coli, yeast, and mammalian cells using orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases (aaRSs) (15,16). These tRNA-aaRS pairs recognize, activate, and incorporate fluorescent and other amino acid analogs into proteins in response to reprogrammed stop codons (17).…”

mentioning

confidence: 99%

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Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

Bezerra

Simões

Lee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

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Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.codon reassignment | evolution | tRNA N atural alterations to the standard genetic code have been discovered in Mycoplasma (1, 2), Micrococci (3), ciliates (4), fungi (5, 6), and mitochondria (7), modifying the hypothesis of a universal genetic code (8). Both neutral (9) and nonneutral theories (10) have been proposed to explain codon reassignments; however, experimental data to support or refute them are scarce, and genetic code alterations remain an intriguing biological puzzle. Despite this fact, it is becoming clear that genetic code alterations are associated with mutations in tRNAs and translation release factors that expand or restrict codon decoding capacity (7). In other words, alterations of translational factors have the potential to release the genetic code from its frozen state. This hypothesis is strongly supported by the widespread cotranslational incorporation of selenocysteine into the active site of selenoprotein (11) and pyrrolysine in the active site of the methyltransferases of several Metanosarcina species (12), Desulfitobacterium hafniense (13), and the gutless worm Olavius algarvensis (14). The selective advantages produced by these two amino acids are associated with evolution of proteins with unique catalytic properties.The flexibility of the genetic code is further highlighted by the in vivo incorporation of artificial amino acids into recombinant proteins of Escherichia coli, yeast, and mammalian cells using orthogonal pairs of tRNA...

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Section: Discussionmentioning

confidence: 34%

mentioning

confidence: 99%

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

Bezerra

Simões

Lee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

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“…Potential structural differences between isoforms IR-A and IR-B (110) are also not well understood. 12 Biophysical effects of the iodo-Tyr B26 modification were recently studied in the context of insulin lispro (containing substitutions Pro B28 3 Lys and Lys B29 3 Pro; the active component of Humalogா (Lilly) (45). Although the structure of the modified analog was not determined, iodo-Tyr B26 was found to mitigate the loss of stability associated with the paired B28 -B29 substitutions.…”

Section: Rigid-body Modeling Distinguished Opposite Edges Of the B26 mentioning

confidence: 99%

“…Given these features, the present study focused on the effects of iodination of Tyr B26 , long known to enhance the affinity of insulin for the IR (7)(8)(9) and recently shown to enhance the pharmaceutical properties of a rapid-acting clinical analog, including its stability and resistance to physical degradation (45). 12 Such findings raise salient questions regarding the role of the iodo-aromatic modification on the structure of the free hormone and its potential role at the hormone-receptor interface. How might iodination of an aromatic residue affect its electrostatic properties and in turn its conformation?…”

Section: -[Iodo-tyrmentioning

confidence: 99%

“…The modification, 3-iodo-Tyr at position B26 (3-I-Tyr B26 ), is associated with enhanced binding to the insulin receptor (IR) (7)(8)(9). The general class of halo-aromatic modifications defines a key frontier of medicinal chemistry (10) and holds promise in the non-standard engineering of proteins through manipulation of systems and weakly polar interactions (11,12). In addition, the -hole of larger halogens can anchor interactions with surrounding polar groups and water molecules (13)(14)(15)(16)(17)(18)(19).…”

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confidence: 99%

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Extending Halogen-based Medicinal Chemistry to Proteins

Hage

Pandyarajan²,

Phillips³

et al. 2016

Journal of Biological Chemistry

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Edited by Norma AllewellInsulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (Tyr B26 ) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum-and molecularmechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-Tyr B26 ]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (Tyr B16 , Phe B24 , Phe B25 , 3-ITyr B26 , and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-Tyr B26 ]insulin analog (determined as an R 6 zinc hexamer). Given that residues B24 -B30 detach from the core on receptor binding, the environment of 3-I-Tyr B26 in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-Tyr B26 engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient -hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins.Insulin, a small protein critical to metabolic homeostasis (1), provides a model for studies of protein folding and design (2) with long-standing application to human therapeutics (3). The hormone contains two chains, A and B (Fig. 1A), linked by two disulfide bridges (cystines A7-B7 and A20 -B19); the A chain is further stabilized by cystine A6 -A11. In pancreatic ␤-cells, insulin is stored within the secretory granules as zinc-coordinated hexamers. This study has exploited insulin semi-synthesis (4) (simplified through the use of norleucine (Nle) 9 at position B29; arrow in Fig. 1A (5)) to investigate a site-specific modification of an aromatic ring by a single halogen atom (6). The modification, 3-iodo-Tyr at position B26 (3-I-Tyr B26 ), is associated with enhanced binding to the insulin receptor (IR)

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Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids

Durkin¹,

Budiša²

2016

Fluorescent Analogs of Biomolecular Building Blocks

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Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli

Cited by 102 publications

References 33 publications

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

Extending Halogen-based Medicinal Chemistry to Proteins

Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids

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