Genetically encoding an electrophilic amino acid for protein stapling and covalent binding to native receptors.

Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology.

Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells.

The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E.

Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids.

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.

Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings.

Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E.