Search

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. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.

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.

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. 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. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)