title: Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.
abstract: We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.
authors: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695 (USA).
authors: Rajendra
authors: R
authors: Uprety
authors: Ji
authors: J
authors: Luo
authors: Jihe
authors: J
authors: Liu
authors: Yuta
authors: Y
authors: Naro
authors: Subhas
authors: S
authors: Samanta
authors: Alexander
authors: A
authors: Deiters
mesh_terms: D002478:Cells, Cultured; D003545:Cysteine / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D005815:Genetic Code / Q000235:genetics*; D057809:HEK293 Cells; D006710:Homocysteine / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D049410:Luciferases, Renilla / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D006710:Homocysteine; D049410:Luciferases, Renilla; D003545:Cysteine
keywords: amino acids; caged compounds; cysteine; gene technology; homocysteine; protein modifications