RESUMO
We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.
Assuntos
Proteínas de Fluorescência Verde/química , Fragmentos de Imunoglobulinas/química , Nanopartículas/química , Anticorpos de Domínio Único/química , Cristalografia por Raios X , DNA/química , Bases de Dados de Proteínas , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , Produtos do Gene gag/química , Células HEK293 , HIV-1/química , Células HeLa , Humanos , Cinética , Ligantes , Microscopia de Fluorescência , Mitose , Domínios Proteicos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/químicaRESUMO
Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.
Assuntos
Corantes Fluorescentes/química , Hidrolases/química , Potenciais de Ação/efeitos dos fármacos , Animais , Bioengenharia , Cálcio/química , Células Cultivadas , Cristalografia por Raios X , Fenômenos Eletrofisiológicos , Corantes Fluorescentes/síntese química , Hidrolases/síntese química , Cinética , Conformação Molecular , Estrutura Molecular , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Proteínas/química , Ratos , RodaminasRESUMO
Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7× increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).
RESUMO
Fluorescent indicators and actuators provide a means to optically observe and perturb dynamic events in living animals. Although chemistry and protein engineering have contributed many useful tools to observe and perturb cells, an emerging strategy is to use chemigenetics: systems in which a small molecule dye interacts with a genetically encoded protein domain. Here we review chemigenetic strategies that have been successfully employed in living animals as photosensitizers for photoablation experiments, fluorescent cell cycle indicators, and fluorescent indicators for studying dynamic biological signals. Although these strategies at times suffer from challenges, e.g. delivery of the small molecule and assembly of the chemigenetic unit in living animals, the advantages of using small molecules with high brightness, low photobleaching, no chromophore maturation time and expanded color palette, combined with the ability to genetically target them to specific cell types, make chemigenetic fluorescent actuators and indicators an attractive strategy for use in living animals.
Assuntos
Corantes Fluorescentes , Engenharia de Proteínas , Animais , Corantes Fluorescentes/química , HumanosRESUMO
Here, we report a method to regulate cellular protein levels by introducing a ubiquitin variant between a destabilizing domain (DD) and the regulated protein. When produced in the absence of a stabilizing ligand the DD dominates and the entire fusion protein is processively degraded by the proteasome. In the presence of the stabilizing ligand the fusion protein is metabolically stable and becomes a substrate for abundant ubiquitin-specific proteases, liberating a native, or a near-native protein-of-interest. This technique is thus particularly useful for the study of proteins whose free N terminus is required for proper function. In addition, removal of the DD in the presence of stabilizing ligand leads to higher expression levels of regulated protein when cells experience transient exposure to a stabilizing ligand, such as in a living animal receiving a single dose of a pharmacological agent as the stabilizing ligand.
Assuntos
Engenharia de Proteínas/métodos , Ubiquitina/metabolismo , Animais , Peptídeo Hidrolases/metabolismo , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/química , Ubiquitina/genéticaRESUMO
G protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. To dissect the function of individual receptor subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely tethered ligands by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates can be used to target a green fluorescent protein-fused metabotropic glutamate receptor by either gene-free application of purified complexes or coexpression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labeling proteins (or suicide enzymes), we set the stage for targeting endogenous receptors in vivo.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Anticorpos de Domínio Único/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ligantes , Processos Fotoquímicos , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Biosensors are used in many fields to measure the concentration of analytes, both in a cellular context and in human samples for medical care. Here, we outline the design of two types of modular biosensors: SNAP-tag-based indicators with a Fluorescent Intramolecular Tether (SNIFITs) and LUCiferase-based Indicators of Drugs (LUCIDs). These semisynthetic biosensors quantitatively measure analyte concentrations in vitro and on cell surfaces by an intramolecular competitive mechanism. We provide an overview of how to design and apply SNIFITs and LUCIDs.
Assuntos
Corantes Fluorescentes/química , Proteínas/genética , Técnicas Biossensoriais/métodos , Linhagem Celular , Escherichia coli/genética , Células HEK293 , Humanos , Engenharia de Proteínas/métodosRESUMO
ß-Hydroxy-α-amino acids are not only used by synthetic chemists but are also found in natural products, many of which show anti-microbial or anti-cancer properties. Over the past 30â years, chemists have searched for many asymmetric routes to these useful building blocks. Initial attempts to synthesize these compounds utilized chiral auxiliaries and the reactions of glycine equivalents with aldehydes to form two stereocenters in one step. Other methods with the formation of specific intermediates or that were aimed at a specific amino acid have also been investigated. Asymmetric hydrogenation by dynamic kinetic resolution has emerged as a high-yielding method for the synthesis of an array of modified amino acids with good stereoselectivity. More recently, amino-acid functionalization and multicomponent reactions have increased the atom economy and simplified many long and difficult routes. In this Focus Review, many of the elegant syntheses of these compounds are explored. The applications of ß-hydroxy-α-amino acids in natural-product synthesis are also mentioned.