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 ** We thank C. Hoppman for helpful discussions, Y.-X. Guo and M. Bowman for help with FPLC purification, J.-K. Weng for pHIS8.3-MBP plasmid, and W. Fischer and W. Low for help with mass spectrometry.

Enhancing protein stability with extended disulfide bonds

“…A survey of the literature reveals that most engineered cysteine disulfides give a Tm increase around 5°C, although there are a few cases where a more than 10°C increase in Tm is observed (27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Interestingly, in work related to that described here, Xiang et al (13) demonstrated that halogen-containing NCAAs with relatively long side chains are capable of forming a covalent linkages with a Cys residue and showed that they improved the thermal stability of an affibody by 13°C in vitro.…”

“…These longer and more flexible linkages should make it much easier to cross-link sites within proteins. Indeed, it has recently been shown that electrophilic amino acids with extended structures can selectively stabilize proteins and protein complexes by intra-and intermolecular cross-links, respectively (13,(23)(24)(25). However, the use of electrophilic NCAAs to form noncanonical cross-links in a bacterial selection system can be complicated by cellular toxicity, irreversible reactions, reaction rates, and scavenging by intracellular nucleophiles.…”

“…This study suggests that an expanded set of amino acid building blocks can provide novel solutions to evolutionary challenges. polyspecific pyrrolysyl-tRNA synthetase termed "XYRS" (13). Based on this work, we designed and synthesized a series of the thiol-containing amino acids SetY, SprY, and SbuY as substrates for XYRS (Fig.…”

“…The calculated length between the two β-carbons of the SbuY-Cys disulfide bond is 14 Å when fully extended, which is significantly longer than a natural cystine cross-link. We genetically encoded these NCAAs in bacterial and mammalian cells by suppressing the nonsense codon TAG with an orthogonal, amber suppressor aminoacyl-tRNA synthetase (RS)/tRNA pair (13). Because statistical analyses of proteins have suggested that stabilizing disulfide mutations are most often found in regions of higher mobility near the protein surface and associated with longer loop lengths (>25 residues) (10,14), we reasoned that these more flexible, extended disulfides might facilitate the introduction of stabilizing disulfide bonds into proteins.…”

Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications

“…One powerful approach for this purpose is (photo-)cross-linking [32,33]. Previous efforts have used tag-mediated labeling [34,35] or unnatural amino acid mutagenesis [36][37][38] to introduce (photo-)cross-linkers to proteins of interest artificially expressed in cells. However, the utility of the (photo-)cross-linking approach should be greatly enhanced if it can be applied to endogenous proteins.…”