RESUMEN
Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.
Asunto(s)
Azidas/química , Química Clic/métodos , Proteínas de Unión al ADN/aislamiento & purificación , Fenilalanina/análogos & derivados , Proteómica/métodos , Factor de Transcripción STAT1/aislamiento & purificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Fenilalanina/química , Fenilalanina/genética , Factor de Transcripción STAT1/química , Factor de Transcripción STAT1/genéticaRESUMEN
A new strategy is described for the modification of CCMV for loading of cargoes inside the viral capsid. Sortase A, an enzyme which is present in Gram-positive bacteria, was used to attach cargo to the glycine-tagged N-termini of several CCMV variants. We show that small molecules and proteins bearing a C-terminal LPETG-motif can be attached in this way. This method allows for the site-specific, covalent, and orthogonal modification of CCMV capsids in a mild fashion, leading to high encapsulation efficiencies. This strategy can easily be expanded to other types of cargoes, labeled with an LPETG-tag without altering protein function.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Bromovirus/química , Cápside/química , Cisteína Endopeptidasas/química , Portadores de Fármacos/química , Modelos Moleculares , Proteínas/administración & dosificación , Proteínas/química , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Strain-promoted oxidation-controlled cyclo-octyne-1,2-quinone cycloaddition (SPOCQ) is a fast and activatable cross-linking strategy for hydrogel formation. Gelation is induced by oxidation, which is performed both chemically using sodium periodate and enzymatically using mushroom tyrosinase. Due to the fast reaction kinetics, SPOCQ-formed hydrogels can be functionalized in one-pot with an azido-containing moiety using SPAAC cross-linking.
RESUMEN
A main challenge in the area of bioconjugation is to devise reactions that are both activatable and fast. Here, we introduce a temporally controlled reaction between cyclooctynes and 1,2-quinones, induced by facile oxidation of 1,2-catechols. This so-called strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) shows a remarkably high reaction rate when performed with bicyclononyne (BCN), outcompeting the well-known cycloaddition of azides and BCN by 3 orders of magnitude, thereby allowing a new level of orthogonality in protein conjugation.
Asunto(s)
Alquinos/química , Catecoles/química , Proteínas/química , Quinonas/química , Azidas/química , Química Clic , Ciclización , Reacción de Cicloadición , Modelos MolecularesRESUMEN
Strain-promoted azide-alkyne cycloaddition (SPAAC) as a conjugation tool has found broad application in material sciences, chemical biology and even in vivo use. However, despite tremendous effort, SPAAC remains fairly slow (0.2-0.5 M(-1) s(-1)) and efforts to increase reaction rates by tailoring of cyclooctyne structure have suffered from a poor trade-off between cyclooctyne reactivity and stability. We here wish to report tremendous acceleration of strain-promoted cycloaddition of an aliphatic cyclooctyne (bicyclo[6.1.0]non-4-yne, BCN) with electron-deficient aryl azides, with reaction rate constants reaching 2.0-2.9 M(-1) s(-1). A remarkable difference in rate constants of aliphatic cyclooctynes versus benzoannulated cyclooctynes is noted, enabling a next level of orthogonality by a judicious choice of azide-cyclooctyne combinations, which is inter alia applied in one-pot three-component protein labelling. The pivotal role of azide electronegativity is explained by density-functional theory calculations and electronic-structure analyses, which indicates an inverse electron-demand mechanism is operative with an aliphatic cyclooctyne.
RESUMEN
Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , Lisina/química , Proteínas/metabolismo , Alquinos/química , Azidas/química , Carbocianinas/química , Química Clic , Cumarinas/química , Reacción de Cicloadición , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Células HeLa , Humanos , Lisina/síntesis química , Microscopía Fluorescente , Ingeniería de Proteínas , Proteínas/química , ARN de Transferencia/metabolismoRESUMEN
The GPCRDB is a Molecular Class-Specific Information System (MCSIS) that collects, combines, validates and disseminates large amounts of heterogeneous data on G protein-coupled receptors (GPCRs). The GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many different types of computationally derived data such as multiple sequence alignments and homology models. The GPCRDB provides access to the data via a number of different access methods. It offers visualization and analysis tools, and a number of query systems. The data is updated automatically on a monthly basis. The GPCRDB can be found online at http://www.gpcr.org/7tm/.