Faculty of 1000 evaluation for Rapid labeling of intracellular His-tagged proteins in living cells (original) (raw)
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Rapid labeling of intracellular His-tagged proteins in living cells
Proceedings of the National Academy of Sciences of the United States of America, 2015
Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhan...
Photoactivatable and Photoconvertible Fluorescent Probes for Protein Labeling
ACS Chemical Biology, 2010
Photosensitive probes are powerful tools to study cellular processes with high temporal and spatial resolution. However, most synthetic fluorophores suited for biomolecular imaging have not been converted yet to appropriate photosensitive analogues. Here we describe a generally applicable strategy for the generation of photoactivatable and photoconvertible fluorescent probes that can be selectively coupled to SNAP-tag fusion proteins in living cells. Photoactivatable versions of fluorescein and Cy3 as well as a photoconvertible Cy5-Cy3 probe were prepared and coupled to selected proteins on the cell surface, in the cytosol, and in the nucleus of cells. In proof-of-principle experiments, the photoactivatable Cy3 probe was used to characterize the mobility of a lipid-anchored cell surface protein and of a G protein coupled receptor (GPCR). This work establishes a generally applicable strategy for the generation of a large variety of different photosensitive fluorophores with tailor-made properties for biomolecular imaging. ARTICLE www.acschemicalbiology.org VOL.5 NO.5 • ACS CHEMICAL BIOLOGY
Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions
Analytical Biochemistry, 2010
The hexahistidine (His 6 )/Nickel (II)-Nitrilotriacetic Acid (Ni 2+ -NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nano-gold to His 6 -proteins. Here we explore several applications of the NTA-derivative, (Ni 2+ -NTA) 2 -Cy3. This molecule binds our two model His 6 -proteins, N-ethylmaleimide Sensitive Factor (NSF) and O 6 -alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 10 6 M -1 ) and no effect on their activity. Its high specificity makes (Ni 2+ -NTA) 2 -Cy3 ideal for detecting His 6 -proteins in complex mixtures of other proteins, allowing (Ni 2+ -NTA) 2 -Cy3 to be used as a probe in crude cell extracts and as a His 6 -specific gel stain. (Ni 2+ -NTA) 2 -Cy3 binding is reversible in 10 mM EDTA or 500 mM imidazole but in their absence, it exchanges slowly (k exchange ∼ 5 × 10 -6 s -1 with 0.2 μM labeled protein in the presence of 1μM His 6 -peptide). Labeling with (Ni 2+ -NTA) 2 -Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions (e.g. high ADP absorbance) that prevent direct detection of protein. In addition, fluorescence resonance energy transfer (FRET) between (Ni 2+ -NTA) 2 -Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.
Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag
Journal of Bacteriology, 2016
The use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryote Escherichia coli. We show that functional protein fusions of the HaloTag can be detected both in vivo and in vitro when expressed within the cytoplasmic or periplasmic compartments of E. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications. IMPORTANCE Visualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryote Escherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.
An Engineered Protein Tag for Multiprotein Labeling in Living Cells
Chemistry & Biology, 2008
The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O 6 -benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIPtag, which reacts specifically with O 2 -benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulsechase experiments to visualize different generations of two different proteins in one sample.
Site-specific Fluorescent Labeling of Poly-histidine Sequences Using a Metal-chelating Cysteine
Chemical Biology & Drug Design, 2007
Coupling genetically encoded target sequences with specific and selective labeling strategies has made it possible to utilize fluorescence spectroscopy in complex mixtures to investigate the structure, function, and dynamics of proteins. Thus, there is a growing need for a repertoire of such labeling approaches to deploy based on a given application and to utilize in combination with one another by orthogonal reactivity. We have developed a simple approach to synthesize a fluorescent probe that binds to a poly-histidine sequence. The amino group of cysteine was converted into nitrilotriacetate to create a metal-chelating cysteine molecule, Cys-nitrilotriacetate. Two Cys-nitrilotriacetate molecules were then cross-linked using dibromobimane to generate a fluorophore capable of binding a His-tag on a protein, NTA 2-BM. NTA 2-BM is a potential fluorophore for selective tagging of proteins in vivo. Keywords cysteine; dibromobimane; fluorescence; His-tag; metal chelating; protein tagging Selective labeling of proteins at defined positions provides a powerful tool to study their structure-function behavior. Protein labeling approaches exploit the chemical reactivity of the side chains of the amino acids and most often use amino-based or thiol-based chemistry. Reactions with labeled proteins are typically monitored using fluorescence spectroscopy, immunodetection, or mass spectrometry. One of the shortcomings of these methods is that labeling is restricted to purified proteins and not appropriate in a mixture of other proteins or in a cellular environment. Recently, there has been intensive effort to develop new probes to site-specifically tag a protein of interest present in a complex biochemical environment. These include the use of fusion constructs with fluorescent proteins (1,2), co-translational introduction of unnatural amino acids or modified amino acids (3-5) or targeting small molecule labels to specific sequences (5-11). The large size of fusion proteins may introduce complexities and limit their use. Moreover, their complex spectral properties can complicate data analysis, for example, distance measurements in fluorescence resonance energy transfer (FRET)-based experiments (12). Incorporation of unnatural amino acids into a growing polypeptide chain using suppressor tRNA technology or 4-base codon methods is one of the
Nano Letters, 2009
Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates trisnitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level.
Fluorescent tags of protein function in living cells
BioEssays, 2000
A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection.