Efforts towards the Design of ‘<i>Teflon</i>’ Proteins: <i>In vivo</i> Translation with Trifluorinated Leucine and Methionine Analogues

Budiša, Nediljko; Pipitone, Olga; Siwanowicz, Igor; Rubini, Marina; Pal, Prajna Paramita; Holak, Tad A.; Gelmi, Maria Luisa

doi:10.1002/cbdv.200490107

Cited by 39 publications

(39 citation statements)

References 35 publications

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“…Recently, Budisa et al (2004) showed the incorporation of trifluorleucine and trifluoromethionine into various proteins over the size of 10 kDa through SPI. Marginal substitution (2-30% by amino acid analysis) was achieved by their method.…”

Section: Discussionmentioning

confidence: 99%

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Panchenko

Zhu

Montclare

2006

Biotechnol. Bioeng.

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Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.

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Section: Discussionmentioning

confidence: 99%

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Panchenko

Zhu

Montclare

2006

Biotechnol. Bioeng.

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“…[36,37] Fluorinated analogues like difluoromethionine (Dfm), trifluoromethio-nine (Tfm) and 6,6,6-trifluoronorleucine (Tfn) have been used as 19 F-NMR probes of protein structure, to increase protein stability and to design teflon-like or "non-stick" proteins. [38][39][40] The more hydrophobic [41] analogues norleucine (Nle) and ethionine (Eth) were used to study overall effects on enzyme activity. Their incorporation in the Met-rich active site of Calmodulin lowered its activity, [42] whereas incorporation of Nle into Lipase from Thermoanaerobacter thermohydrosulfuricus (TTL) led to an enzyme highly active in the aqueous phase without the need for thermal activation [43] and enhanced stability against denaturing agents.…”

Section: Global Reassignment Of the Aug Codonmentioning

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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“…In hindsight, this is not surprising as a CF 3 group should be ~70% bulkier than CH 3 (C-F bond length 1.39 Å vs. C-H 1.09 Å; van der Waals radii of 1.35 Å for F vs. 1.2 Å for H), and may be more hydrophobic as suggested by partitioning studies of hexafluoroleucine and leucine 48 (also see ref. 42 and references therein).…”

Section: Selecting a New Trna Synthetase For A Fluorinated Unnatural mentioning

confidence: 99%

In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance Spectroscopy

et al. 2008

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In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by co-expressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy) phenyl) propanoic acid (OCF 3 Phe), 13 C/ 15 N-labeled p-methoxyphenylalanine (OMePhe), and 15 N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3 Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of E. coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in 1 H-15 N, 1 H-13 C HSQC and 19 F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3 Phe mutants of an active site tyrosine inhibited binding; incorporating 15 N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

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Efforts towards the Design of ‘Teflon’ Proteins: In vivo Translation with Trifluorinated Leucine and Methionine Analogues

Cited by 39 publications

References 35 publications

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

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

In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance Spectroscopy

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