Site‐specific Protein Cross‐Linking with Genetically Incorporated 3,4‐Dihydroxy‐<scp>L</scp>‐Phenylalanine

Umeda, Aiko; Thibodeaux, Gabrielle Nina; Zhu, Jie; Lee, YungAh; Zhang, Zhiwen Jonathan

doi:10.1002/cbic.200900127

Cited by 28 publications

(21 citation statements)

References 14 publications

(22 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[18] In another experiment, all the Met residues in the model protein green fluorescent protein (GFP) were replaced with the "clickable" ncAAs Aha or Hpg, whereas the photo-crosslinker 3,4-dihydroxyphenylalanine (L-DOPA) was incorporated at a specific site. [19] Recent improvements in the SCS methodology, i.e. evolving more efficient aaRSs in a genomically recoded E. coli strain, have greatly increased the suppression efficiency allowing the multi-site incorporation of a single ncAA at up to 30 positions in protein-polymers with high yields.…”

Section: Classical Methods For In Vivo Multiplementioning

confidence: 99%

“…Labeling of proteins with selenomethionine (SeMet) and telluromethionine (TeMet) has become a common practice for structure determination by X-ray crystallography. [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.…”

Section: Global Reassignment Of the Aug Codonmentioning

confidence: 99%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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.

show abstract

Section: Classical Methods For In Vivo Multiplementioning

confidence: 99%

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

View full text Add to dashboard Cite

show abstract

“…While there have been several successful demonstrations of the rational placement of crosslinking amino acids into proteins (Alfonta et al, 2003; Forne et al, 2012; Sato et al, 2011; Umeda et al, 2009, 2010), to date there has not been a thorough analysis of how a detailed structural analysis might be used to guide the placement of amino acids that would crosslink efficiently. Currently, placement of NCAAs into proteins is based on intuition and no computational tools exist to guide the design of crosslinks.…”

Section: Discussionmentioning

confidence: 99%

“…l -DOPA has previously been used to successfully crosslink the monomeric domains of a dimeric sortase A for structural studies (Umeda et al, 2009), to enhance the affinity of low-affinity peptide probes for a kinase SH3 bioassay (Umeda et al, 2010), and to site-specifically label proteins with polysaccharides (Ayyadurai et al, 2011). While previously reported uses of l -DOPA as a site-specific crosslinker have yielded examples of effective crosslinking (as shown by SDS–PAGE or Western blot analyses), the actual efficiencies of crosslinking have never been reported (Burdine et al, 2004; Umeda et al, 2009, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…While previously reported uses of l -DOPA as a site-specific crosslinker have yielded examples of effective crosslinking (as shown by SDS–PAGE or Western blot analyses), the actual efficiencies of crosslinking have never been reported (Burdine et al, 2004; Umeda et al, 2009, 2010). These previous reports indicated that it was possible to place l -DOPA by intuition, but did not provide more quantitative assessments of what parameters impacted crosslinking efficiency (Umeda et al, 2009, 2010). …”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Tack

Hughes

et al. 2014

Journal of Structural Biology

View full text Add to dashboard Cite

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.

show abstract

Development of a Selective, Sensitive, and Reversible Biosensor by the Genetic Incorporation of a Metal‐Binding Site into Green Fluorescent Protein

Ayyadurai¹,

Prabhu²,

Deepankumar³

et al. 2011

Angewandte Chemie

View full text Add to dashboard Cite

Site‐specific Protein Cross‐Linking with Genetically Incorporated 3,4‐Dihydroxy‐L‐Phenylalanine

Cited by 28 publications

References 14 publications

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

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

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Development of a Selective, Sensitive, and Reversible Biosensor by the Genetic Incorporation of a Metal‐Binding Site into Green Fluorescent Protein

Contact Info

Product

Resources

About