Structural Insights into Incorporation of Norbornene Amino Acids for Click Modification of Proteins

Schneider, Sabine; Gattner, Michael; Vrábel, Milan; Flügel, Veronika; López‐Carrillo, Verónica; Prill, Stefan; Carell, Thomas

doi:10.1002/cbic.201300435

Cited by 37 publications

(34 citation statements)

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“…Structural analyses have also revealed how mutant PylRS enzymes can accommodate ncAAs that are chemically distinct (50) and larger than Pyl (47), including furan-containing Lys (46) and norbornene-containing Pyl analogs (51). Larger ncAAs are accepted by these PylRS variants by creating larger amino acid recognition pockets with mutations to smaller active site residues, particularly at position 306.…”

Section: Resultsmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

104

146

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Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

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Section: Resultsmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

104

146

View full text Add to dashboard Cite

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“…This opens the door to many applications of these probes in proteomics or in the synthesis of irreversible inhibitors of some DNA-processing enzymes. [50] In the past few years, severalm ethods have been developedt oi ntroduce unnatural amino acids (UAA) into proteins. Mutant R273H is not trappedb yDNA 2EVS (Figure 2c, lane 9), since the proximity effect is crucial for cross-linking.…”

Section: Resultsmentioning

confidence: 99%

“…To verify its potentiali n protein modification, human carbonic anhydrase II (hCA)-containing terminal acetylene in as pecific position was selected as am odel protein. [50] The modified hCA (hCA*)w as expressed in Escherichia coli by using the amber codon suppression and orthogonal pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase system (Figure 3). [51] One of the most powerful approaches for incorporating UAA site-specifically into proteins expressed in cells is geneticc ode expansion.…”

Section: Resultsmentioning

confidence: 99%

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA–Protein Cross‐Linking

Daďová

Vrábel

Adámik

et al. 2015

Chemistry A European J

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N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

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“…10c, 16 The norbornene functionality was incorporated at positions Asn65 of Trx (Trx N65X) and His36 of HCA (HCA H36X) (for expression and purification details see the ESI †). We next synthesized sulfonyl azide derivatives 5 and 6 bearing a biotin tag or a dansyl fluorophore to examine the norbornene aziridination on proteins (Fig.…”

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confidence: 99%

Sulfonyl azide-mediated norbornene aziridination for orthogonal peptide and protein labeling

2014

Self Cite

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We describe a new bioconjugation reaction based on the aziridination of norbornenes using electron-deficient sulfonyl azides. The reaction enables to attach various useful tags to peptides and proteins under mild conditions.Bioconjugation reactions substantially extend our ability to chemically manipulate proteins. Numerous chemical strategies for the attachment of synthetic molecules to proteins have been developed.1 Early approaches focused on native functional groups present on endogenous amino acids. 2 The main drawback of this approach is the lack of specificity since multiple copies of each amino acid are present in the primary protein structure. Unique recognition elements can be introduced into the protein structure to improve the selectivity of the ligation. One possibility is to add a specific amino acid sequence to the target protein that can be recognized by either an appropriate protein modifying enzyme 3 or by specific chemical probes. 4Another approach uses the biosynthetic machinery of the cell for incorporation of unnatural amino acids containing artificial functional groups. 5 We and others have used this approach for the incorporation of unnatural amino acids into proteins and have shown that the introduced functionality can be efficiently tagged by orthogonal chemical reactions. 6 The right choice of the reacting functional groups and the proper ligation technique plays a crucial role here. 7 Among other suitable functional groups that enable efficient protein labeling various derivatives of cyclooctyne, cyclooctene and cyclopropene have gained special attention in the field. 8 An extraordinary fast kinetic was observed especially in combination with tetrazines in inverse electrondemand Diels-Alder reactions or in dipolar cycloadditions with nitrile imines. 8a,b,e Unfortunately, the high reactivity of such systems is often accompanied by reduced stability and an increased tendency toward side reactions. 9 Moreover, synthetic access to these reagents often represents a considerable challenge. It is therefore desirable to develop a methodology that not only enables robust protein labeling but also utilizes easily available starting materials. Based on our experience using norbornenes as highly reactive compounds in combination with nitrile oxides, nitrile imines and tetrazines 10 we searched for reagents that do react with norbornenes, but also meet the criteria for better synthetic accessibility. Here we show that aziridination of norbornenes using electron-deficient sulfonyl azides represents an excellent balance between reactivity, stability and availability of the starting materials. We demonstrate that this reaction can be used for efficient labeling of norbornenecontaining peptides and proteins. Inspired by the pioneering studies on norbornene aziridination by Franz et al.,11 we decided to investigate whether this reaction can proceed in an aqueous environment and can be used for biomolecule labeling. Sulfonyl azides have been previously successfully applied as reagents for detectin...

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Structural Insights into Incorporation of Norbornene Amino Acids for Click Modification of Proteins

Cited by 37 publications

References 100 publications

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA–Protein Cross‐Linking

Sulfonyl azide-mediated norbornene aziridination for orthogonal peptide and protein labeling

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