Unnatural amino acid mutagenesis-based enzyme engineering

Ravikumar, Yuvaraj; Nadarajan, Sivakumar; Yoo, Tae Hyeon; Lee, Chong‐Soon

doi:10.1016/j.tibtech.2015.05.002

Cited by 72 publications

(48 citation statements)

References 102 publications

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“…[1,2] On the other hand, ncAAs which are orthogonal to the host cell translation system are usually incorporated sitespecifically by suppression of stop codons (SCS) using orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pairs (o-pairs). [3] Although the incorporation of one single ncAA into proteins has provided fascinating insights into sequencefunction relationships [2][3][4] as well as potential applications in enzymology, [5][6][7] it is often desirable to introduce simultaneously different chemical functionalities by using two or more ncAAs. [8,9] For example, Budisa and coworkers introduced three ncAAs in parallel into the model protein barstar using the SPI method: [10] the incorporation of the Trp analogue 4-azatryptophan (4AzaTrp) endowed the protein with a blue fluorescent probe for bio-imaging, [11] the Met analogue homopropargylglycine (Hpg) equipped it with a biorthogonal handle for post-translational conjugation by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), [12] while the Pro analogue (4S)-FPro ((4S)-FPro) provided a stabilizing effect on its structure.…”

Section: Classical Methods For In Vivo Multiplementioning

confidence: 99%

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNA fMet initiates protein synthesis (starting AUG codon), whereas elongator tRNA Met participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNA Met pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNA Met from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNA Pyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

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Section: Classical Methods For In Vivo Multiplementioning

confidence: 99%

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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“…The incorporation of non‐coded amino acids into proteins has emerged as a promising approach in protein science for elucidating the molecular mechanisms underlying protein folding, stability, and function . Compared to peptide engineering, protein engineering with non‐coded amino acids has a greater predictive power of the mutational effects.…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, an unnatural amino acid, added to the cell growth medium, is specifically recognized by a modified aaRS and coupled to an amber suppressor tRNA, which is decoded by the ribosome in response to an amber codon (UAG) introduced into the gene of interest, thus allowing the synthesis of a protein with a site‐specifically introduced unnatural amino acid . Genetic code expansion techniques allowed the site‐specific incorporation into different protein systems of more than 100 diverse non‐coded amino acids, carrying novel chemical entities and spectroscopic probes, for studying protein/enzyme structure, folding, stability, and function . Although whole‐cell expression methods have much improved the overall efficiency of mutagenesis with non‐coded amino acids, compared to cell‐free methods originally introduced , genetic methods are still limited by the low amount of the resulting mutant proteins that can be obtained …”

Section: Introductionmentioning

confidence: 99%

“…Genetic code expansion techniques allowed the site‐specific incorporation into different protein systems of more than 100 diverse non‐coded amino acids, carrying novel chemical entities and spectroscopic probes, for studying protein/enzyme structure, folding, stability, and function . Although whole‐cell expression methods have much improved the overall efficiency of mutagenesis with non‐coded amino acids, compared to cell‐free methods originally introduced , genetic methods are still limited by the low amount of the resulting mutant proteins that can be obtained …”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Protein engineering by chemical methods: Incorporation of nonnatural amino acids as a tool for studying protein folding, stability, and function

et al. 2018

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Proteins are large complex biomolecules that act as the effectors of essentially all cell functions. Due to the intrinsic complexity of protein architecture at the microscopic level and the inadequacy of theoretical methods to predict protein reactivity (ie, folding, stability, and function), protein engineering has emerged as a valuable tool to investigate structure–stability–activity relationships in proteins and nowadays recombinant DNA technologies are the “gold standard” for site‐specifically manipulating a given protein chain. The usefulness of current mutagenesis techniques, however, is limited by the relatively poor chemical diversity of the 20 DNA‐coded amino acids, such that it is difficult to precisely assign the observed change of protein stability or function to the variation of a single physicochemical property at a protein site (ie, hydrophobicity, conformational propensity, polarizability, hydrogen bonding, etc). In this article, we report relevant examples from our laboratory showing that chemical methods, that is, enzyme‐catalyzed semisynthesis and stepwise solid‐phase synthesis, allow to conveniently incorporate non‐natural amino acids with “tailored” side chains into small proteins and thus effectively transfer the structure–activity relationship methodology, typical of the medicinal chemistry approach on small molecules, to the study of folding, stability, and molecular recognition in macromolecular protein systems.

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“…2) The auxotrophic E. coli K12 derivative W3110 ( Ec E3110) strain used is a good choice because it is genetically stable (the complete Trp operon is removed, in contrast to other Trp‐auxotrophic strains). 3) The synthetic route for [3,2]Tpa excludes any indole or Trp derivative that could contaminate commercial preparations of Trp analogues (e.g., <0.03 % in the case of 4FW), thus avoiding cellular scavenging that would result in an inefficient ncAA incorporation. 4) The simple addition of [3,2]Tp to the medium suffices because, like indole, it can diffuse across the cell membrane and be converted into [3,2]Tpa if an active Trp‐synthase enzyme (TrpBA) is present (Trp is synthesised from indole and serine in living cells).…”

Section: Methodsmentioning

confidence: 99%