RESUMO
Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.
Assuntos
Biologia Computacional/métodos , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Ligases/metabolismo , Oxazinas/metabolismo , Coloração e Rotulagem , Animais , Biocatálise , Células COS , Sobrevivência Celular , Chlorocebus aethiops , Cumarínicos , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Imageamento Tridimensional , Microscopia Eletrônica , Modelos Moleculares , Mutagênese , Oxazinas/síntese química , Oxazinas/química , RatosRESUMO
Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.
Assuntos
Azidas/metabolismo , Ciclo-Octanos/metabolismo , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Ligases/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Azidas/química , Biocatálise , Células COS , Células Cultivadas , Chlorocebus aethiops , Ciclização , Ciclo-Octanos/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Corantes Fluorescentes/química , Células HEK293 , Células HeLa , Humanos , Ligases/química , Ligases/genética , Proteínas Luminescentes/química , Estrutura Molecular , MutaçãoAssuntos
Fármacos Fotossensibilizantes/química , Bloqueadores dos Canais de Potássio/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Isomerismo , Ligantes , Estrutura Molecular , Técnicas de Patch-Clamp , Fármacos Fotossensibilizantes/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Relação Estrutura-AtividadeRESUMO
This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.
Assuntos
Proteínas de Membrana/química , Coloração e Rotulagem/métodos , Alcinos/química , Azidas/química , Quelantes , Cobre/química , Reação de Cicloadição/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Ligases/genética , Proteínas de Membrana/análise , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately 'blind' neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3ß4 and α4ß2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.