Proximity‐Enabled Protein Crosslinking through Genetically Encoding Haloalkane Unnatural Amino Acids

Abstract

The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity-enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity-enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering.

Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency

“…This increase in the thermal stability could be due to more covalent bonds between the monomers of crosslinked C1 enzyme than WT enzyme according to Xiang et al . 36 .

Figure 7SDS-PAGE analysis of SrtA-mediated ligation of 8 oxidoreductases after incubating for 36 h. Mono-oxidoreductases included oxidoreductase-mtf, oxidoreductase-GGGGSLPET and cyclized oxidoreductase-GGGGSLPET. Ligation products, mainly dimers and trimers, were labelled as crosslinked oxidoreductases.

Figure 8Thermal shift curves of 8 oxidoreductases and their crosslinked oxidoreductases.

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Genetically encoding new bioreactivity

“…A series of haloalkane Uaas were designed and synthesized containing different halogen atoms linked with aliphatic chains of varying length (Figure 4B) [46]. The M. mazei ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}∕\text{PylRs}$ pair was evolved to incorporate these Phe-scaffold Uaas [32] into myoglobin in E. coli and mammalian cells.…”

“…ESI-FTMS analysis of mutant myoglobin showed that the Uaas were specifically incorporated and no side reaction of the Uaa with Cys, glutathione or any other amino acid residue was observed, indicating that the Uaa did not undergo modification or nonspecific self-reaction. These haloalkane Uaas, after incorporation into the affibody, react with a proximal Cys of the Z protein at the interface, crosslinking the two proteins irreversibly in the efficiency of ~45% in 1h [46]. Similarly, the Cl-PSCaa, 2-amino-3-(4-((3-(chloromethyl)phenyl)diazenyl)phenyl)propanoic acid, with benzyl chloride installed on an azobenzene was shown to be incorporated by the evolved ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ pair in E. coli and mammalian cells, and the incorporated Cl-PSCaa reacts with a proximal Cys to form an intramolecular photoswitchable bridge (Figure 4C) [47].…”

“…The ability to genetically encode a bioreactive Uaa selectively reacting with a natural amino acid residue under mild conditions now enables the specific introduction of covalent bonds for proteins in vitro and in live cells, which we call Proximity Enabled Protein Crosslinking (PEPC) [34,46]. PEPC can occur within or between proteins to afford new opportunities for protein research and engineering.…”

“…Haloalkane Uaa and Cys were introduced into the same Afb protein to build a covalent protein bridge crosslinking its two α–helices (Figure 5B) [46]. After protein expression in E. coli followed by purification, mutant proteins of Afb-30BetY-47C and Afb-30BprY-47C clearly showed strong peaks with monoisotopic masses corresponding to the Afb containing a covalent Cys-Uaa bond at the introduced positions, indicating successful intra-molecular crosslinking.…”