RESUMEN
We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl-tRNA synthetase/tRNACUA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off-to-on light switching. This reporter was then used to evaluate the activation rates of the different light-removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox.
Asunto(s)
Endopeptidasas/metabolismo , Endopeptidasas/efectos de la radiación , Luz , Procesos Fotoquímicos/efectos de la radiación , Ingeniería de Proteínas , Tirosina/análogos & derivados , Tirosina/genética , Endopeptidasas/química , Endopeptidasas/genética , Código Genético , Tirosina/metabolismo , Tirosina/efectos de la radiaciónRESUMEN
A number of non-canonical amino acids (NCAAs) with unstrained olefins are genetically encoded using mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pairs. These NCAAs readily undergo inverse electron-demand Diels-Alder cycloadditions with tetrazine dyes, leading to selective labeling of proteins bearing these NCAAs in live cells.
Asunto(s)
Alquenos/química , Colorantes Fluorescentes/química , Compuestos Heterocíclicos con 1 Anillo/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Reacción de Cicloadición , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fluoresceína/química , Proteínas Fluorescentes Verdes/química , Hidrolasas/química , Hidrolasas/metabolismo , Mutación , ARN de Transferencia/metabolismoRESUMEN
Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylTCUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins.
Asunto(s)
Alquenos/metabolismo , Multimerización de Proteína , Compuestos de Sulfhidrilo/metabolismo , Animales , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Lisina/metabolismo , Methanosarcina barkeri/metabolismo , Muramidasa/metabolismo , Mioglobina/metabolismoRESUMEN
Detailed kinetic analyses of inverse electron-demand DielsAlder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these "click" reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.
Asunto(s)
Alquenos/química , Alquinos/química , Química Clic , Proteínas/química , Coloración y Etiquetado/métodos , Catálisis , Ciclooctanos/química , Proteínas de Escherichia coli/química , Cinética , Modelos Moleculares , Conformación Proteica , Tirosina/químicaRESUMEN
We report the first site-specific genetic encoding of photocaged tyrosine into proteins in mammalian cells. By photocaging Tyr701 of STAT1 we demonstrate that it is possible to photocontrol tyrosine phosphorylation and signal transduction in mammalian cells.
Asunto(s)
Luz , Ingeniería de Proteínas , Factor de Transcripción STAT1/metabolismo , Tirosina/metabolismo , Aminoacil-ARNt Sintetasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Methanosarcina/enzimología , Methanosarcina/genética , Mutación , Fosforilación , Ingeniería de Proteínas/métodos , Factor de Transcripción STAT1/genética , Transducción de Señal , Tirosina/genéticaRESUMEN
The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Norbornanos/química , Proteínas/metabolismo , Aminoácidos/metabolismo , Línea Celular , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Proteínas/genética , Rodaminas/químicaRESUMEN
Microwave irradiation has been proven to be a useful tool in the rapid assembly of racemic unnatural amino acids in only two steps. Additional benefits of this methodology are the commercial availability of the inexpensive starting materials and the high yields and high purities of the final amino acid products.