Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains

Abstract

The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein-protein interactions and improve the pharmacological properties of proteins. Here we report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra- or intermolecular protein crosslinks. We evolved a pyrrolysyl-tRNA synthetase that can site-specifically incorporate PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification; crosslinking required incubation under weakly basic condition. We demonstrated that FPheK can react with adjacent Lys, Cys and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab. The high efficiency and specificity of FPheK crosslinking may make it a practical tool to engineer proteins with unnatural covalent linkages.

Expanding the Genetic Code to Study Protein–Protein Interactions

“…[35] Proximity-triggered protein crosslinking obviates the need for an external trigger for reactivity and affords residue specificity,and it has recently emerged as ap owerful tool for stabilizing lowaffinity protein complexes to aid their structural elucidation and for capturing transient protein-protein interactions in living cells.U AAs with electrophilic moieties that undergo proximity-enabled crosslinking that can be incorporated sitespecifically into proteins include reactive halides, [36] Michael acceptors, [37] aryl isothiocyanates, [38] aryl carbamates, [39] and aryl fluorosulfates (Figure 7). [35] Proximity-triggered protein crosslinking obviates the need for an external trigger for reactivity and affords residue specificity,and it has recently emerged as ap owerful tool for stabilizing lowaffinity protein complexes to aid their structural elucidation and for capturing transient protein-protein interactions in living cells.U AAs with electrophilic moieties that undergo proximity-enabled crosslinking that can be incorporated sitespecifically into proteins include reactive halides, [36] Michael acceptors, [37] aryl isothiocyanates, [38] aryl carbamates, [39] and aryl fluorosulfates (Figure 7).…”

“…Kurzaufsätze predominantly in increasing the effective local concentration of the reactants,with effective molar concentrations reaching values as high as 10 5 m and consequently increasing reaction rates and enabling reactions that would not yield products in the absence of the concentration effect. [35] Proximity-triggered protein crosslinking obviates the need for an external trigger for reactivity and affords residue specificity,and it has recently emerged as ap owerful tool for stabilizing lowaffinity protein complexes to aid their structural elucidation and for capturing transient protein-protein interactions in living cells.U AAs with electrophilic moieties that undergo proximity-enabled crosslinking that can be incorporated sitespecifically into proteins include reactive halides, [36] Michael acceptors, [37] aryl isothiocyanates, [38] aryl carbamates, [39] and aryl fluorosulfates (Figure 7). [40]…”

“…[36c] The bromoalkyl moiety is not only reactive towards proximal cysteines,but also forms covalent linkages with nearby lysine and histidine residues in in vitro crosslinking experiments under basic conditions (pH 8.8). Apart from showing proofof-principle reactivity,b yc rosslinking known affibody/Zprotein complexes in vitro,U AA 17 (n = 5) was used to irreversibly bind aH ER2-specific affibody to its native human epidermal growth factor receptor 2( HER2/Erb2) on the surface of breast cancer cells.T oe xpand chemical crosslinking reactivity toward other nucleophilic amino acid residues besides cysteines,f urther UAAs bearing vinylsulfonamide (18), [37] aryl isothiocyanate (19), [38] aryl carbamate (20), [39] and aryl fluorosulfate (21) [40] moieties were designed. These UAAs show crosslinking reactivities in in vitro experiments,p rimarily towards lysine residues,a sw ell as towards histidine (21)a nd tyrosine (20, 21), but they often require basic conditions (pH > 8.5), which restricts their application to in vitro protein crosslinking experiments.…”

Site-Specific Incorporation of a Thioester Containing Amino Acid into Proteins

“…Multiple rounds of positive and negative selection in E. coli DH10B were performed in the presence of 2 mM ThioD via the published method. 25 One colony was isolated that survived two positive and one negative selection; DNA sequencing revealed a PylRS variant (ThioDRS, Figure 2B) harboring the mutations Ala267Tyr, Tyr271Ala, Asn311Thr, Cys313Gly and Tyr349Phe. Analysis of these mutations in ThioDRS reveals a strong rationale for its recognition of ThioD - the Asn311Thr mutation appears to make space for the thioester group while the Cys313Gly and Tyr271Ala mutations make the cavity larger to accommodate the hydrophobic cyclopentane ring; the Ala267Tyr mutation may result in a hydrogen bond to the carbamate group.…”

Synthesis of poly(Nε‐phenoxycarbonyl‐l‐lysine) by polycondensation of activated urethane derivative and its application for selective modification of side chain with amines

“…N ε‐Phenoxycarbonyl moieties are known to be moderate electrophiles, and are available to react with amines to produce the corresponding urea derivatives. By taking advantage of the highly efficient reaction of its aryl carbamate, Schultz and coworker reported some crosslinking applications with protein molecules, suggesting that the strategy is compatible with a wide array of biologics . To test whether that modification of side chains is possible for poly‐Lys(NPC), we examined different types of model amines (A1‐A6) including n ‐BuNH 2 , and 2‐amino ethanol as the primary amine, aniline as an aromatic amine, diallylamine as the secondary amine, and L‐alanine methyl ester as an amino acid derivative for post‐polymerization modification (Fig.…”