Expanding the genetic code with a lysine derivative bearing an enzymatically removable phenylacetyl group

Reille-Seroussi, Marie; Mayer, Susanne V.; Dörner, Wolfgang; Lang, Kathrin; Mootz, Henning D.

doi:10.1039/c9cc00475k

Cited by 9 publications

(12 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Of note, the rates and efficiencies observed here for PGA‐mediated deprotection reveal an unexpectedly high catalytic potential on protein substrates. They represent a dramatic improvement over previously reported PGA deprotection schemes of Lys(Pac) or Cys(Phacm) on both peptide [12, 13, 16] and protein [13a, 17] substrates, which were often incomplete and required overnight incubation. Next to the structural dependencies, it should be noted that PGA has typically been used as a commercial sample from industrial production batches, often in immobilized form.…”

Section: Resultsmentioning

confidence: 85%

“…Next to the structural dependencies, it should be noted that PGA has typically been used as a commercial sample from industrial production batches, often in immobilized form. We recommend not to use such samples for protein applications due to potential problems with protein degradation and precipitation, likely due to their crude nature [13a] …”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

et al. 2021

Self Cite

View full text Add to dashboard Cite

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.

show abstract

Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The accessibility of the side chain of 1 should depend on the globular fold around its position. [13a] In our initial deprotection assays (Figure 1 ), 1 was located close to the N terminus of an unstructured tail region, thus exhibiting nearly maximal accessibility. We systematically tested other positions with varying distance from the N terminus in diSUMO model proteins (Figures 2 B and S4–S6) and analyzed time‐dependent deprotection by ESI‐MS as described above.…”

Section: Resultsmentioning

confidence: 95%

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mass spectrometry : MS analysis of intact proteins and an in‐gel tryptic digest of the splice product band was carried out as previously described [33] . For mass spectrometry of intact proteins from splice assays the used elution gradient (5–60 % acetonitrile) was applied over 45 min.…”

Section: Methodsmentioning

confidence: 99%

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

et al. 2020

Self Cite

View full text Add to dashboard Cite

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of nonredundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

show abstract

Expanding the genetic code with a lysine derivative bearing an enzymatically removable phenylacetyl group

Abstract: Site-specific incorporation of a lysine analog with an enzymatically removable side chain protection group was used to control protein activity.

Cited by 9 publications

References 37 publications

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

Contact Info

Product

Resources

About