title: Directed evolution of ligand dependence: small-molecule-activated protein splicing.
abstract: Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Allen R
authors.initials: AR
authors.lastname: Buskirk
authors.forename: Yi-Ching
authors.initials: YC
authors.lastname: Ong
authors.forename: Zev J
authors.initials: ZJ
authors.lastname: Gartner
authors.forename: David R
authors.initials: DR
authors.lastname: Liu
mesh_terms: D000563:Amebicides / Q000494:pharmacology; D019020:Directed Molecular Evolution; D004965:Estrogen Antagonists / Q000494:pharmacology; D005839:Gentamicins / Q000494:pharmacology; D008024:Ligands; D008958:Models, Molecular; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000563:Amebicides; D004965:Estrogen Antagonists; D005839:Gentamicins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013629:Tamoxifen; C016601:afimoxifene; C010680:antibiotic G 418