1. Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian Cells
- Author
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Hoppmann, Christian, Lacey, Vanessa K, Louie, Gordon V, Wei, Jing, Noel, Joseph P, and Wang, Lei
- Subjects
Chemical Sciences ,Generic health relevance ,Amino Acids ,Click Chemistry ,Escherichia coli ,Genetic Code ,Molecular Conformation ,Optogenetics ,Protein Engineering ,amino acids ,click chemistry ,isomerization ,optogenetics ,photoswitches ,Organic Chemistry ,Chemical sciences - Abstract
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.
- Published
- 2014