NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.
TL;DR: The shortened FAST is designed, which is composed of only 98 amino acids, the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, by truncating 26 N-terminal residues.
Abstract: One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.
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TL;DR: In this paper, a collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design.
Abstract: Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Forster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.
21 citations
TL;DR: In this article, the authors highlight the pros and cons of a wide variety of transiently interacting labels and discuss the state of the art and future perspectives of low-affinity labeling methods.
Abstract: Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them—the requirement for high photostability—can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.
7 citations
TL;DR: A comprehensive understanding of effector translocation in a spatio-temporal manner is of critical importance to gain insights into an effector’s mode of action.
Abstract: Bacteria-host interactions are characterized by the delivery of bacterial virulence factors, i.e., effectors, into host cells where they counteract host immunity and exploit host responses allowing bacterial survival and spreading. These effectors are translocated into host cells by means of dedicated secretion systems such as the type 3 secretion system (T3SS). A comprehensive understanding of effector translocation in a spatio-temporal manner is of critical importance to gain insights into an effector’s mode of action. Various approaches have been developed to understand timing and order of effector translocation, quantities of translocated effectors and their subcellular localization upon translocation into host cells. Recently, the existing toolset has been expanded by newly developed state-of-the art methods to monitor bacterial effector translocation and dynamics. In this review, we elaborate on reported methods and discuss recent advances and shortcomings in this area of tracking bacterial effector translocation.
6 citations
TL;DR: In this paper, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated.
Abstract: Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.
3 citations
TL;DR: In this paper , the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen, was described, and two mutants appeared brighter than the wild-type FAST, and these mutants provided up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo.
Abstract: "Fluorescence-Activating and absorption-Shifting Tag" (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.
3 citations
References
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TL;DR: The utility of this modular protein tagging system for cellular imaging and protein immobilization is demonstrated by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.
Abstract: We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-κB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein−protein and protein−DNA complexes.
1,822 citations
TL;DR: A general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalentlabeling of proteins and that may open up new ways of studying proteins in living cells is described.
Abstract: Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.
1,702 citations
TL;DR: The structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging are focused on, with particular attention to recent techniques.
Abstract: Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.
1,214 citations
TL;DR: The generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP provide an approach for genetic encoding of fluorescent RNAs and create a palette that spans the visible spectrum.
Abstract: Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.
1,111 citations
TL;DR: In this article, a monomeric yellow green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum, was described.
Abstract: We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
1,043 citations