Chemical tags for labeling proteins inside living cells

To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.     

Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

Visualizing biochemical activities in living cells through chemistry

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.     

A Dual Small‐Molecule Rheostat for Precise Control of Protein Concentration in Mammalian Cells

One of the most successful strategies for controlling protein concentrations in living cells relies on protein destabilization domains (DD). Under normal conditions, a DD will be rapidly degraded by the proteasome. However, the same DD can be stabilized or “shielded” in a stoichiometric complex with a small molecule, enabling dose‐dependent control of its concentration. This process has been exploited by several labs to post‐translationally control the expression levels of proteins in vitro as well as in vivo, although the previous technologies resulted in permanent fusion of the protein of interest to the DD, which can affect biological activity and complicate results. We previously reported a complementary strategy, termed traceless shielding (TShld), in which the protein of interest is released in its native form. Here, we describe an optimized protein concentration control system, TTShld, which retains the traceless features of TShld but utilizes two tiers of small molecule control to set protein concentrations in living cells. These experiments provide the first protein concentration control system that results in both a wide range of protein concentrations and proteins free from engineered fusion constructs. The TTShld system has a greatly improved dynamic range compared to our previously reported system, and the traceless feature is attractive for elucidation of the consequences of protein concentration in cell biology.     

Development of Benzophenone‐Alkyne Bifunctional Sigma Receptor Ligands

Sigma (σ) receptors are unique non‐opioid binding sites that are associated with a broad range of disease states. Sigma‐2 receptors provide a promising target for diagnostic imaging and pharmacological interventions to curb tumor progression. Most recently, the progesterone receptor (PGRMC1, 25 kDa) has been shown to have σ2 receptor‐like binding properties, thus highlighting the need to understand the biological function of an 18 kDa protein that exhibits σ2‐like photoaffinity labeling (denoted here as σ2‐18k) but the amino acid sequence of which is not known. In order to provide new tools for the study of the σ2‐18k protein, we have developed bifunctional σ receptor ligands each bearing a benzophenone photo‐crosslinking moiety and an alkyne group to which an azide‐containing biotin affinity tag can be covalently attached through click chemistry after photo‐crosslinking. Although several compounds showed favorable σ2 binding properties, the highest affinity (2 nM ) and the greatest potency in blocking photolabeling of σ2‐18k by a radioactive photoaffinity ligand was shown by compound 22 . These benzophenone‐alkyne σ receptor ligands might therefore be amenable for studying the σ2‐18k protein through chemical biology approaches. To the best of our knowledge, these compounds represent the first reported benzophenone‐containing clickable σ receptor ligands, which might potentially have broad applications based on the “plugging in” of various tags.     

Polyamide Fluorescent Probes for Visualization of Repeated DNA Sequences in Living Cells

DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole‐imidazole polyamides that bind specifically to the minor groove of double‐stranded DNA (dsDNA) represent an attractive approach for in‐cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel‐shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.     

Recent Developments in Metal-Catalyzed Bio-orthogonal Reactions for Biomolecule Tagging

With the rapid advances in single-molecule and live-cell imaging studies to investigate biological problems, the role of chemical probes to monitor reactions in a live cell has considerably increased. However, selective labeling of a target protein or a specific residue is highly challenging due to the high complexity of the biological system. In particular, biological macromolecules (such as proteins, DNA, or RNA) share many functional groups that potentially cross-react with exogenous chemical probes. Thus, there are high demands for perfect biocompatible reactions utilizing biologically unavailable chemistry. Metal-catalyzed reactions have been extensively investigated as synthetic methodology studies, including initial attempts in applying the chemistry in aqueous solutions in vitro or even in biological conditions. Herein, the latest developments and progress in metal-catalyzed bio-orthogonal reactions for biomolecule labeling are summarized.     

Introducing bioorthogonal functionalities into proteins in living cells

Proteins are the workhorses of the cell, playing crucial roles in virtually every biological process. The revolutionary ability to visualize and monitor proteins in living systems, which is largely the result of the development of green fluorescence protein (GFP) and its derivatives, has dramatically expanded our understanding of protein dynamics and function. Still, GFPs are ill suited in many circumstances; one major drawback is their relatively large size, which can significantly perturb the functions of the native proteins to which they are fused. To bridge this gap, scientists working at the chemistry-biology interface have developed methods to install bioorthogonal functional groups into proteins in living cells. The bioorthogonal group is, by definition, a non-native and nonperturbing chemical group. But more importantly, the installed bioorthogonal handle is able to react with a probe bearing a complementary functionality in a highly selective fashion and with the cell operating in its physiological state. Although extensive efforts have been directed toward the development of bioorthogonal chemical reactions, introducing chemical functionalities into proteins in living systems remains an ongoing challenge. In this Account, we survey recent progress in this area, focusing on a genetic code expansion approach. In nature, a cell uses posttranslational modifications to append the necessary functional groups into proteins that are beyond those contained in the canonical 20 amino acids. Taking lessons from nature, scientists have chosen or engineered certain enzymes to modify target proteins with chemical handles. Alternatively, one can use the cell's translational machinery to genetically encode bioorthogonal functionalities, typically in the form of unnatural amino acids (UAAs), into proteins; this can be done in a residue-specific or a site-specific manner. For studying protein dynamics and function in living cells, site-specific modification by means of genetic code expansion is usually favored. A variety of UAAs bearing bioorthogonal groups as well as other functionalities have been genetically encoded into proteins of interest. Although this approach is well established in bacteria, tagging proteins in mammalian cells is challenging. A facile pyrrolysine-based system, which might potentially become the "one-stop shop" for protein modification in both prokaryotic and eukaryotic cells, has recently emerged. This technology can effectively introduce a series of bioorthogonal handles into proteins in mammalian cells for subsequent chemical conjugation with small-molecule probes. Moreover, the method may provide more precise protein labeling than GFP tagging. These advancements build the foundation for studying more complex cellular processes, such as the dynamics of important receptors on living mammalian cell surfaces.     

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution ¹⁹F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.     

Chemoselectivity in chemical biology: acyl transfer reactions with sulfur and selenium

A critical source of insight into biological function is derived from the chemist's ability to create new covalent bonds between molecules, whether they are endogenous or exogenous to a biological system. A daunting impediment to selective bond formation, however, is the myriad of reactive functionalities present in biological milieu. The high reactivity of the most abundant molecule in biology, water, makes the challenges all the more difficult. We have met these challenges by exploiting the reactivity of sulfur and selenium in acyl transfer reactions. The reactivity of both sulfur and selenium is high compared with that of their chalcogen congener, oxygen. In this Account, we highlight recent developments in this arena, emphasizing contributions from our laboratory. One focus of our research is furthering the chemistry of native chemical ligation (NCL) and expressed protein ligation (EPL), two related processes that enable the synthesis and semisynthesis of proteins. These techniques exploit the lower pK(a) of thiols and selenols relative to alcohols. Although a deprotonated hydroxyl group in the side chain of a serine residue is exceedingly rare in a biological context, the pK(a) values of the thiol in cysteine (8.5) and of the selenol in selenocysteine (5.7) often render these side chains anionic under physiological conditions. NCL and EPL take advantage of the high nucleophilicity of the thiolate as well as its utility as a leaving group, and we have expanded the scope of these methods to include selenocysteine. Although the genetic code limits the components of natural proteins to 20 or so α-amino acids, NCL and EPL enable the semisynthetic incorporation of a limitless variety of nonnatural modules into proteins. These modules are enabling chemical biologists to interrogate protein structure and function with unprecedented precision. We are also pursuing the further development of the traceless Staudinger ligation, through which a phosphinothioester and azide form an amide. We first reported this chemical ligation method, which leaves no residual atoms in the product, in 2000. Our progress in effecting the reaction in water, without an organic cosolvent, was an important step in the expansion of its utility. Moreover, we have developed the traceless Staudinger reaction as a means for immobilizing proteins on a solid support, providing a general method of fabricating microarrays that display proteins in a uniform orientation. Along with NCL and EPL, the traceless Staudinger ligation has made proteins more readily accessible targets for chemical synthesis and semisynthesis. The underlying acyl transfer reactions with sulfur and selenium provide an efficient means to synthesize, remodel, and immobilize proteins, and they have enabled us to interrogate biological systems.     

Phosphoproteomics and Lung Cancer Research

Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed.     

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

Classical fluorescence‐based approaches to monitor ligand–protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn‐on probes for a G protein‐coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor‐specific turn‐on response. The new ligand was successfully applied for background‐free imaging and quantification of oxytocin receptors in living cells.     

Bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins

Understanding protein structure and function is essential for uncovering the secrets of biology, but it remains extremely challenging because of the high complexity of protein networks and their wiring. The daunting task of elucidating these interconnections requires the concerted application of methods emerging from different disciplines. Chemical biology integrates chemistry, biology, and pharmacology and has provided novel techniques and approaches to the investigation of biological processes. Among these, site-specific protein labeling with functional groups such as fluorophors, spin probes, and affinity tags has greatly facilitated both in vitro and in vivo studies of protein structure and function. Bioorthogonal chemical reactions, which enable chemo- and regioselective attachment of small-molecule probes to proteins, are particularly attractive and relevant for site-specific protein labeling. The introduction of powerful labeling techniques also has inspired the development of novel strategies for surface immobilization of proteins to create protein biochips for in vitro characterization of biochemical activities or interactions between proteins. Because this process requires the efficient immobilization of proteins on surfaces while maintaining structure and activity, tailored methods for protein immobilization based on bioorthogonal chemical reactions are in high demand. In this Account, we summarize recent developments and applications of site-specific protein labeling and surface immobilization of proteins, with a special focus on our contributions to these fields. We begin with the Staudinger ligation, which involves the formation of a stable amide bond after the reaction of a preinstalled azide with a triaryl phosphine reagent. We then examine the Diels-Alder reaction, which requires the protein of interest to be functionalized with a diene, enabling conjugation to a variety of dienophiles under physiological conditions. In the oxime ligation, an oxyamine is condensed with either an aldehyde or a ketone to form an oxime; we successfully pursued the inverse of the standard technique by attaching the oxyamine, rather than the aldehyde, to the protein. The click sulfonamide reaction, which involves the Cu(I)-catalyzed reaction of sulfonylazides with terminal alkynes, is then discussed. Finally, we consider in detail the photochemical thiol-ene reaction, in which a thiol adds to an ene group after free radical initiation. Each of these methods has been successfully developed as a bioorthogonal transformation for oriented protein immobilization on chips and for site-specific protein labeling under physiological conditions. Despite the tremendous progress in developing such transformations over the past decade, however, the demand for new bioorthogonal methods with improved kinetics and selectivities remains high.     

Techniques for monitoring protein misfolding and aggregation in vitro and in living cells

Protein misfolding and aggregation have been considered important in understanding many neurodegenerative diseases and recombinant biopharmaceutical production. Various traditional and modern techniques have been utilized to monitor protein aggregation in vitro and in living cells. Fibril formation, morphology and secondary structure content of amyloidogenic proteins in vitro have been monitored by molecular probes, TEM/AFM, and CD/FTIR analyses, respectively. Protein aggregation in living cells has been qualitatively or quantitatively monitored by numerous molecular folding reporters based on either fluorescent protein or enzyme. Aggregation of a target protein is directly correlated to the changes in fluorescence or enzyme activity of the folding reporter fused to the target protein, which allows non-invasive monitoring aggregation of the target protein in living cells. Advances in the techniques used to monitor protein aggregation in vitro and in living cells have greatly facilitated the understanding of the molecular mechanism of amyloidogenic protein aggregation associated with neurodegenerative diseases, optimizing culture conditions to reduce aggregation of biopharmaceuticals expressed in living cells, and screening of small molecule libraries in the search for protein aggregation inhibitors.     

From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants

The study of protein–protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.     

Fatty Acyl Sulfonyl Fluoride as an Activity-Based Probe for Profiling Fatty Acid-Associated Proteins in Living Cells

Fatty acids play fundamental structural, metabolic, functional, and signaling roles in all biological systems. Altered fatty acid levels and metabolism have been associated with many pathological conditions. Chemical probes have greatly facilitated biological studies on fatty acids. Herein, we report the development and characterization of an alkynyl-functionalized long-chain fatty acid-based sulfonyl fluoride probe for covalent labelling, enrichment, and identification of fatty acid-associated proteins in living cells. Our quantitative chemical proteomics show that this sulfonyl fluoride probe targets diverse classes of fatty acid-associated proteins including many metabolic serine hydrolases that are known to be involved in fatty acid metabolism and modification. We further validate that the probe covalently modifies the catalytically or functionally essential serine or tyrosine residues of its target proteins and enables evaluation of their inhibitors. The sulfonyl fluoride-based chemical probe thus represents a new tool for profiling the expression and activity of fatty acid-associated proteins in living cells.     

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.