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Cellular activities are predominantly carried out by proteins that can dynamically adopt different structural conformations and differentially interact with other biomolecules according to cellular needs. Chemical probes are small molecules used to selectively interact and modulate the activities of specific proteins to study their functions such as the validation of potential drug targets. The remarkable performance of AlphaFold algorithms in the prediction of protein structures has pivoted interest toward elucidating the intracellular dynamics of protein structural conformation where covalent modification of proteins by chemical probes could be used to shed light upon. However, due to the barrier to entry by cell membrane and the general unfavorable reactive conditions of the intracellular environment, most studies using reactive chemical probes are still conducted on purified proteins and cell lysates. Nevertheless, recent progresses have been made in designing chemical probes with improved membrane permeability, stability and reactivity. This paper surveys the literature on recent advancements in membrane-permeable chemical probes and their applications with protein mass spectrometry for the intracellular studies of protein structural conformations and biomolecular interactions., (© 2024 Wiley-VCH GmbH.)

Reversible bioorthogonal conjugation reactions have been exploited in the chemoproteomic field to prepare protein labeling reagents and to visualize labeled proteins. We recently demonstrated that reversible iminoboronates can be used to prepare probes from fragment libraries and that the linkage subsequently can be used to detect the labeled proteins. In this study, we determined the effect of the stability of the iminoboronate linkage on the efficiency of the labeling protocol. Our study reveals that the linkage should be stable enough to allow for efficient targeting, but should be labile enough to detect the labeled protein. Acyl hydrazides were identified as the most suitable handles for the probe synthesis step. Anthranilic hydrazides and N -hydroxy semicarbazides were found to be the most efficient read-out molecules. With these novel exchange molecules, native probe-labeled proteins could be visualized under physiological conditions.

Protein glycation is a disease associated, non-enzymatic, posttranslational modification generated by endogenous dicarbonyl metabolites. Currently, there is a lack of chemical tools capable of studying protein adducts caused by this class of reactive species. Here, we report a chemical biology platform, termed T-DiP (targetable-dicarbonyl precursor), that releases a physiologically relevant dose of bio-orthogonally functionalized dicarbonyl probe upon irradiation with 365 nm light. This approach enables protein glycation to be controlled with spatiotemporal precision within live cells and expands the chemical toolbox needed to elucidate the roles of glycated proteins across various pathologies.

Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of "inducible" activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations.

The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site-specific incorporation of unnatural amino acids in proteins, and post-synthetic labelling of nucleic acids. While a majority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets., (© 2021 Wiley-VCH GmbH.)

Abstact: The growing prevalence of synthetically modified proteins in pharmaceuticals and materials has exposed the need for efficient strategies to enable chemical modifications with high site-selectivity. While genetic engineering can incorporate non-natural amino acids into recombinant proteins, regioselective chemical modification of wild-type proteins remains a challenge. Herein, we use photoredox catalysis to develop a site-selective tyrosine bioconjugation pathway that incorporates bioorthogonal formyl groups, which subsequently allows for the synthesis of structurally defined fluorescent conjugates from native proteins. A water-soluble photocatalyst, lumiflavin, has been shown to induce oxidative coupling between a previously unreported phenoxazine dialdehyde tag and a single tyrosine site, even in the presence of multiple tyrosyl side chains, through the formation of a covalent C-N bond. A variety of native proteins, including those with multiple tyrosines, can successfully undergo both tyrosine-specific and single-site-selective labelling. This technology directly introduces aldehyde moieties onto native proteins, enabling rapid product diversification using an array of well-established bioorthogonal functionalization protocols including the alkyne-azide click reaction., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE. For complete details on the use and execution of these protocols, please refer to Corley et al. (2020)., Competing Interests: G.W.Y. is co-founder, member of the Board of Directors, equity holder, and paid consultant for Locana and Eclipse Bioinnovations. G.W.Y. is a Distinguished Visiting Professor at the National University of Singapore. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies. M.C. and G.W.Y. are inventors on a patent disclosure to UCSD on technologies described in this manuscript. H.Y.C. is an inventor on a patent owned by Stanford University on in vivo SHAPE. H.Y.C. is a co-founder of Accent Therapeutics and Boundless Bio and an advisor for 10× Genomics, Arsenal Biosciences, and Spring Discovery. H.Y.C. is an Investigator of the Howard Hughes Medical Institute., (© 2021.)

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable 'turn-on' fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.

High-quality small molecule chemical probes are extremely valuable for biological research and target validation. However, frequent use of flawed small-molecule inhibitors produces misleading results and diminishes the robustness of biomedical research. Several public resources are available to facilitate assessment and selection of better chemical probes for specific protein targets. Here, we review chemical probe resources, discuss their current strengths and limitations, and make recommendations for further improvements. Expert review resources provide in-depth analysis but currently cover only a limited portion of the liganded proteome. Computational resources encompass more proteins and are regularly updated, but have limitations in data availability and curation. We show how biomedical scientists may use these resources to choose the best available chemical probes for their research.

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni 2+ -NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni 2+ -NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAP Switch ) that can be conjugated to biomolecules using click chemistry. SNAP Switch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAP Switch , we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAP Switch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

Targeted protein degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the ubiquitin-proteasome system. Over the last 5 years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine., Competing Interests: Declaration of Interests C.M.C. is founder, shareholder, and consultant to Arvinas, Inc. and Halda, LLC, which support research in his laboratory., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. Recognition that the traditional rules of probe discovery are not necessarily applicable to dynamic proteins and their complexes, as well as technological advances in screening, have produced remarkable results in the last 2-4 years. Particularly notable are the improvements in target selectivity for small-molecule modulators of dynamic proteins, especially with techniques that increase the discovery likelihood of allosteric regulatory mechanisms. We focus on approaches to small-molecule screening that appear to be more suitable for highly dynamic targets and have the potential to streamline identification of selective modulators., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Activity-based protein profiling (ABPP) has become an emerging chemical proteomic approach to illustrate the interaction mechanisms between compounds and proteins. This approach has combined organic synthesis, biochemistry, cell biology, biophysics and bioinformatics to accelerate the process of drug discovery in target identification and validation, as well as in the stage of lead discovery and optimization. This review will summarize new developments and applications of ABPP in medicinal chemistry. Here, we mainly described the design principles of activity-base probes (ABPs) and general workflows of ABPP approach. Moreover, we discussed various basic and advanced ABPP strategies and their applications in medicinal chemistry, including competitive and comparative ABPP, two-step ABPP, fluorescence polarization ABPP (FluoPol-ABPP) and ABPs for visualization. In conclusion, this review will give a general overview of the applications of ABPP as a powerful and efficient technique in medicinal chemistry., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Perfluorinated organic compounds (PFCs) are nontoxic, biocompatible, bioavailable, and bioorthogonal species which possess the unique ability to segregate away from both polar and nonpolar solvents producing a compact fluorophilic phase. Traditional techniques of fluorous chemical proteomics are generally applied to enrich biological samples in target protein(s) exploiting this property of PFCs to build fluorinated probes able to covalently bind to protein ensembles and being selectively extracted by fluorophilic solvents. Aiming at building a strategy able to avoid irreversible modification of the analyzed biosystem, a novel fully noncovalent probe is presented as an enabling tool for the recognition and isolation of biological protein(s). In our strategy, both the fluorophilic extraction and the biorecognition of a selected protein successfully occur via the establishment of reversible but selective interactions.

For most immunoblots developed with chemiluminescence or with fluorochrome-based detection systems, it is possible to remove the primary and secondary antibodies from the membrane without affecting the bound antigen. This allows you to reuse the membrane for detection of another protein antigen. The blots developed with chromogenic substrates can also be stripped of antibodies and reprobed, but the bands detected in the first round of immunoblotting will remain unaffected. Stripping and reprobing of the membrane are particularly useful when the amount of sample is limited or when it is important to accurately compare the signal between two different protein antigens in the same sample. Examples of such experiments include determining the levels of a protein antigen in a series of samples relative to the loading control and comparison of the phosphorylated form to the total levels of the protein in the sample., (© 2020 Cold Spring Harbor Laboratory Press.)

Oxidative modifications of cysteine thiols in cellular proteins are pivotal to the way signal-stimulated reactive oxygen species are sensed and elicit appropriate or sometimes pathological responses, but the dynamic and often transitory nature of these modifications offer a challenge to the investigator trying to identify such sites and the responses they elicit. A number of reagents and workflows have been developed to identify proteins undergoing oxidation and to query the timing, extent and location of such modifications, as described in this minireview. While no approach is perfect to capture all the redox information in a functioning cell, best practices described herein can enable considerable insights into the "redox world" of cells and organisms., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Lysine residues have been considered as a routine conjugating site for protein chemical labeling and modification. The commercially available lysine-labeling agents have several limitations in labeling efficiency, stability, and cost. To pursue alternative protein lysine-labeling strategies, herein, we report the development of an ortho -phthalaldehyde (OPA)-based bifunctional linker suitable for protein chemical labeling and profiling. Among three designed OPA-based bifunctional linkers, OPA-NH-alkyne 5 was proved to be optimal for protein labeling with minimal protein turbidity. We further demonstrated OPA-NH-alkyne 5 was applicable for immediate capture of protein or proteome chemical labeling.

We developed a new method for the de novo formation of fluorophores based on citrate (DNFC) in biological samples. Use of an amide coupling reagent and microwave irradiation greatly facilitates the fluorophore formation on peptides and proteins with N-terminal cysteine or serine. Since N-terminal cysteine and serine can form thiazolopyridone- or oxazolopyridone-based fluorophores emitting blue and green fluorescence, respectively, by the DNFC staining, each organelle, cell and tissue exhibited a characteristic fluorescence distribution. The DNFC staining is able to provide a new potential protocol for future cell imaging, histology and diagnosis.

A fundamental challenge in chemical biology and medicine is to understand and expand the fraction of the human proteome that can be targeted by small molecules. We recently described a strategy that integrates fragment-based ligand discovery with chemical proteomics to furnish global portraits of reversible small-molecule/protein interactions in human cells. Excavating clear structure-activity relationships from these 'ligandability' maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments. Here, we describe a compelling solution to this problem by introducing a next-generation set of fully functionalized fragments differing only in absolute stereochemistry. Using these enantiomeric probe pairs, or 'enantioprobes', we identify numerous stereoselective protein-fragment interactions in cells and show that these interactions occur at functional sites on proteins from diverse classes. Our findings thus indicate that incorporating chirality into fully functionalized fragment libraries provides a robust and streamlined method to discover ligandable proteins in cells.

The plethora of methods developed for the creation of protein conjugates often differs significantly with regard to the heterogeneity of the resulting products, in the degree of genetic manipulation of the protein required, and in the technical skills required to perform the conjugation procedure. Affinity-guided protein conjugation is a protein labeling methodology based on noncovalent binding interactions between a labeling probe and the protein of interest. These interactions increase the local concentration of a reactive group in the probe on the protein surface thus facilitating the conjugation in proximity of the complexation site. The ability to produce high-quality conjugates from nongenetically modified proteins both in vitro, but also in cells, demonstrates the power of affinity-guided protein conjugation. Here, we present the progress of affinity-guided protein conjugation in relation to selective protein labeling in living systems and the formation of high-quality protein conjugates. Furthermore, the probe design will be discussed in relation to the utility of the probe for labeling in vitro or in living systems., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemical labeling of proteins with synthetic low-molecular-weight probes is an important technique in chemical biology. To achieve this, it is necessary to use chemical reactions that proceed rapidly under physiological conditions (i.e., aqueous solvent, pH, low concentration, and low temperature) so that protein denaturation does not occur. The radical reaction satisfies such demands of protein labeling, and protein labeling using the biomimetic radical reaction has recently attracted attention. The biomimetic radical reaction enables selective labeling of the C-terminus, tyrosine, and tryptophan, which is difficult to achieve with conventional electrophilic protein labeling. In addition, as the radical reaction proceeds selectively in close proximity to the catalyst, it can be applied to the analysis of protein-protein interactions. In this review, recent trends in protein labeling using biomimetic radical reactions are discussed., Competing Interests: The authors declare no conflict of interest.

Inducing biomolecular interactions with synthetic molecules to impact biological function is a concept of enormous appeal. Recent years have seen a resurgence of interest in designing bispecific molecules that serve as bridging agents to bring proteins together. Pioneering structural and biophysical investigation of ternary complexes formed by mono-functional and bifunctional ligands highlights that proximity-induced stabilization or de novo formation of protein-protein interactions is a common feature of their molecular recognition. In this review, we illustrate these concepts and advances with representative case studies, and highlight progress over the past three years, with particular focus on recruitment to E3 ubiquitin ligases by 'molecular glues' and chimeric dimerizers (PROTACs) for targeted protein degradation. This approach promises to significantly expand the range of tractable targets for chemical biology and therapeutic intervention., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The function of enzymatic proteins is given by their ability to bind specific small molecules into their active sites. These sites can often be found in pockets on a hypothetical boundary between the protein and its environment. Detection, analysis, and visualization of pockets find its use in protein engineering and drug discovery. Many definitions of pockets and algorithms for their computation have been proposed. Kawabata and Go defined them as the regions of empty space into which a small spherical probe can enter but a large probe cannot and developed programs that can compute their approximate shape. In this article, this definition was slightly modified in order to capture the existence of large internal holes, and a Voronoi-based method for the computation of the exact shape of these modified regions is introduced. The method first puts a finite number of large probes on the protein exterior surface and then, considering both large probes and atomic balls as obstacles for the small probe, the method computes the exact shape of the regions for the small probe. This is all achieved with Voronoi diagrams, which help with the safe navigation of spherical probes among spherical obstacles. Detected regions are internally represented as graphs of vertices and edges describing possible movements of the center of the small probe on Voronoi edges. The surface bounding each region is obtained from this representation and used for visualization, volume estimation, and comparison with other approaches. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Bioorthogonal reactions that proceed readily under physiological conditions without interference from biomolecules have found widespread application in the life sciences. Complementary to the bioorthogonal reactions that ligate two molecules, reactions that release a molecule or cleave a linker are increasingly attracting interest. Such dissociative bioorthogonal reactions have a broad spectrum of uses, for example, in controlling bio-macromolecule activity, in drug delivery, and in diagnostic assays. This review article summarizes the developed bioorthogonal reactions linked to a release step, outlines representative areas of the applications of such reactions, and discusses aspects that require further improvement., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High-resolution NMR spectroscopy is sensitive to local structural variations and subtle dynamics of biomolecules and is an important technique for studying the structures, dynamics, and interactions of these molecules. Small-molecule probes, including paramagnetic tags, have been developed for this purpose. Paramagnetic effects manifested in magnetic resonance spectra have long been recognized as valuable tools for chemical analysis of small molecules, and these effects were later applied in the fields of chemical biology and structural biology. However, such applications require the installation of a paramagnetic center in the biomolecules of interest. Paramagnetic metal ions and stable free radicals are the most widely used paramagnetic probes for biological magnetic resonance spectroscopy, and therefore mild, high-yielding approaches for chemically attaching paramagnetic tags to biomolecules are in high demand. In this Account, we begin by discussing paramagnetic species, especially transition metal ions and lanthanide ions, that are suitable for NMR and EPR studies, particularly for in-cell applications. Thereafter, we describe approaches for site-specific tagging of proteins with paramagnetic ions and discuss considerations involved in designing high-quality paramagnetic tags, including the strength of the binding between the metal-chelating moiety and the paramagnetic ion, the chemical stability, and the flexibility of the tether between the paramagnetic tag and the target protein. The flexibility of a tag correlates strongly with the averaging of paramagnetic effects observed in NMR spectra, and we describe methods for increasing tag rigidity and applications of such tags in biological systems. We also describe specific applications of established site-specific tagging approaches and newly developed paramagnetic tags for the elucidation of protein structures and dynamics at atomic resolution both in solution and in cells. First, we describe the determination of the 3D structure of a short-lived, low-abundance enzyme intermediate complex in real time by using pseudocontact shifts as structural restraints. Second, we demonstrate the utility of stable paramagnetic tags for determining 3D structures of proteins in live cells, and pseudocontact shifts are shown to be valuable structural restraints for in-cell protein analysis. Third, we show that a NMR optimized paramagnetic tag allows one to determine distance restraints on proteins by double electron-electron resonance (DEER) measurements with high spatial resolution both in vitro and in cells. Finally, we summarize recent advances in site-specific tagging of proteins to achieve atomic-resolution information about structural changes of proteins, and the advantages and challenges of magnetic resonance spectroscopy in biological systems.

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.

Labeling of a protein with a specific dye or tag at defined positions is a critical step in tracing the subtle behavior of the protein and assessing its cellular function. Over the last decade, many strategies have been developed to achieve selective labeling of proteins in living cells. In particular, the site-specific unnatural amino acid (UAA) incorporation technique has gained increasing attention since it enables attachment of various organic probes to a specific position of a protein in a more precise way. In this review, we describe how the UAA incorporation technique has expanded our ability to achieve site-specific labeling and visualization of target proteins for functional analyses in live cells.

Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M -1 s -1 and k = 5.99 M -1 s -1 , respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

Chemical modification of proteins provides powerful tools to realize a broad range of exciting biological applications, including the development of new classes of biopharmaceuticals and functional studies of individual proteins in complex biological systems. Numerous strategies for linking desired chemical probes with target proteins have been developed in the last two decades, with most exploiting genetic protein engineering and/or bio-orthogonal chemistry that utilizes unnatural amino acids incorporated into proteins. Modification of native proteins in test tubes and biological contexts by site-specific and target-selective approaches remains challenging because appropriate organic chemistry to carry out such modifications is currently limited. Nonetheless, a variety of promising strategies have appeared recently that address this grand challenge in chemical biology. These new chemistries yield native protein-based well-defined bioconjugations, specific labeling of endogenous proteins in various biological crude milieus, and the establishment of chemical proteomics as a new research area in protein science. In this Perspective, we focus on recent remarkable progress in chemistry for native protein modification. We survey chemical characteristics of the methods and describe briefly these advanced applications to address unsolved biological issues. Current limitations and future directions of this research field are also discussed.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins (BCPs) have been the subject of extensive research over the past decade, resulting in a plethora of high-quality chemical probes for their tandem bromodomains. In turn, these chemical probes have helped reveal the profound biological role of the BET bromodomains and their role in disease, ultimately leading to a number of molecules in active clinical development. However, the BET subfamily represents just 8/61 of the known human bromodomains, and attention has now expanded to the biological role of the remaining 53 non-BET bromodomains. Rapid growth of this research area has been accompanied by a greater understanding of the requirements for an effective bromodomain chemical probe and has led to a number of new non-BET bromodomain chemical probes being developed. Advances since December 2015 are discussed, highlighting the strengths/caveats of each molecule, and the value they add toward validating the non-BET bromodomains as tractable therapeutic targets., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NMR spectroscopy is particularly adept at site-specifically monitoring dynamic processes in proteins, such as protein folding, domain movements, ligand binding, and side-chain rotations. By coupling the favorable spectroscopic properties of highly dynamic side-chain methyl groups with transverse-relaxation-optimized spectroscopy (TROSY), it is now possible to routinely study such dynamic processes in high-molecular-weight proteins and complexes approaching 1 MDa. In this Perspective, we describe many elegant methyl-based NMR experiments that probe slow (second) to fast (picosecond) dynamics in large systems. To demonstrate the power of these methods, we also provide interesting examples of studies that utilized each methyl-based NMR technique to uncover functionally important dynamics. In many cases, the NMR experiments are paired with site-directed mutagenesis and/or other biochemical assays to put the dynamics and function into context. Our vision of the future of structural biology involves pairing methyl-based NMR spectroscopy with biochemical studies to advance our knowledge of the motions large proteins and macromolecular complexes use to choreograph complex functions. Such studies will be essential in elucidating the critical structural dynamics that underlie function and characterizing alterations in these processes that can lead to human disease.

O-linked N-acetylglucosamine ( O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.

Many intracellular proteins are metabolically unstable, and their half-life was known to be controlled by the "N-end rule," that is, the N-terminal residue controlled protein stability. To visualize or measure the cellular stability of a protein, depending on the N-terminal residues, attention is being paid to the development of selective labeling methods for individual N-terminal amino acids. However, there are only a limited number of functional groups available for specific N-terminal amino acid labeling in a biological environment. Herein, we report a re-examination of salicylaldehyde ester for selective N-terminal residue tagging. Salicylaldehyde ester has been used for chemical ligation to N-terminal serine or threonine under pyridine/acetic acid conditions. Inspired by previous selective serine/threonine labeling, N-terminal labeling of salicylaldehyde ester in aqueous buffer has been examined by using boron-dipyrromethene (BODIPY), rhodamine, and coumarin probes. Surprisingly, the selectivity not only significantly differed, depending on the fluorophore incorporated in salicylaldehyde, but was also perturbed by the addition of a small fraction of phosphate-buffered saline. In particular, the coumarin-based salicylaldehyde ester probe showed notable selectivity against N-terminal cysteine under aqueous buffer conditions. This result reveals the serendipitous discovery of a new N-terminal cysteine labeling strategy., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of small-molecule fluorophores to label proteins with minimal perturbation in response to an external stimulus is a powerful tool to probe chemical and biochemical environments. Herein, we describe the use of a coumarin-modified triazabutadiene that can deliver aryl diazonium ions to fluorescently label proteins by tyrosine-selective modification. The labeling can be triggered by low-pH-induced liberation of the diazonium species, thus making the fluorophore especially useful in labeling biochemical surroundings such as those found within the late endosome. Additionally, we show that a variety of coumarin triazabutadienes might also be prone to releasing their diazonium cargo after irradiation with UV light., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazoles. A low potential is applied in an electrochemical cell to activate urazole anchors in situ and on demand, without affecting the electroactive amino acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin, and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.

Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Studies on the human proteome have engaged diverse techniques; however, none of them represent a predominant approach. Chemical biology has made a major contribution to our understanding of human biology, stimulating the generation of biological hypotheses. Tools such as functional probes have advanced studies on biological mechanisms and helped in elucidating off-target reactivity and potential toxicities of drugs and drug candidates. Here, we accentuate the recent developments in the design and applications of phosph(on)ate-containing probes. Phosphate esters and anhydrides are present in a number of vital cell constituents, and their significance can be reflected by a number of biological processes that involve phosphorus-bearing molecules. We discuss the use of phosph(on)ate-derived probes for (1) the identification of phosphate-requiring enzymes, their substrates, interacting partners; (2) developing screening assays; and (3) their potential as diagnostics. Limitations that as yet need to be overcome and possible measures to be undertaken will also be addressed.

Chemical probes are of central relevance for chemical biology. To unambiguously explore the role of target proteins in triggering or mediating biological functions, small molecules used as probes should ideally be target-specific; at least, they should have sufficiently high selectivity for a primary target. We present a thorough analysis of currently available activity data for designated chemical probes to address several key questions: How well defined are chemical probes? What is their level of selectivity? Is there evidence for additional activities? Are some probes "better" than others? Therefore, highly curated chemical probes were collected and their selectivity was analyzed on the basis of publicly available compound activity data. Different selectivity patterns were observed, which distinguished designated high-quality probes., Competing Interests: The authors declare no conflict of interest.

Protein conjugates of high heterogeneity may contain species with significantly different biological properties, and as a consequence, the focus on methods for production of conjugates of higher quality has increased. Here, we demonstrate an efficient and generic approach for the modification of metal-binding proteins with biocompatible chemical handles without the need for genetic modifications. Affinity-guided small-molecule probes are developed for direct conjugation to off-the-shelf proteins and for installing different chemical handles on the protein surface. While purification of protein conjugates obtained by small molecule conjugation is troublesome, the affinity motifs of the probes presented here allow for purification of the conjugates. The versatility of the probes is demonstrated by conjugation to several His-tagged and natural metal-binding proteins, including the efficient and area-selective conjugation to three therapeutically relevant antibodies.

The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by nonspecific interactions with off-target species. Here, we describe and demonstrate an assay that utilizes both the chemical and physical properties of the target species to achieve high selectivity in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nanocircuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilization or washing steps are required, and our assay is well suited to monoepitopic targets, presenting advantages over conventional ELISA techniques.

Mapping proteins at a specific subcellular location is essential to gaining detailed insight on local protein dynamics. We have developed an enzymatic strategy to label proteins on a subcellular level using arylamine N-acetyltransferase (NAT). The NAT enzyme activates an arylhydroxamic acid functionality into a nitrenium ion that reacts fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. The electron density on the aromatic ring proved important for probe activation as strong labeling was only observed with an arylhydroxamic acid bearing an electron donating substituent. We further demonstrate that, using this electron rich arylhydroxamic acid, clear labeling was achieved on a subcellular level in living cells that were transfected with a genetically targeted NAT to the nucleus or the cytosol.

Mixed-solvent molecular dynamics (MixMD) is a cosolvent simulation technique for identifying binding hotspots and specific favorable interactions on a protein's surface. MixMD studies have the ability to identify these biologically relevant sites by examining the occupancy of the cosolvent over the course of the simulation. However, previous MixMD analysis required a great deal of manual inspection to identify relevant sites. To address this limitation, we have developed MixMD Probeview as a plugin for the freely available, open-source version of the molecular visualization program PyMOL. MixMD Probeview incorporates two analysis procedures: (1) identifying and ranking whole binding sites and (2) identifying and ranking local maxima for each probe type. These functionalities were validated using four common benchmark proteins, including two with both active and allosteric sites. In addition, three different cosolvent procedures were compared to examine the impact of including more than one cosolvent in the simulations. For all systems tested, MixMD Probeview successfully identified known active and allosteric sites based on the total occupancy of neutral probe molecules. As an easy-to-use PyMOL plugin, we expect that MixMD Probeview will facilitate identification and analysis of binding sites from cosolvent simulations performed on a wide range of systems.

We report a strategy to introduce a reactive electrophile into proteins through the conversion of a chemically inert group into a bioreactive group in response to an inducer molecule. This strategy was demonstrated by oxidation-induced and proximity-enhanced protein-protein crosslinking in the presence of a large excess of free nucleophile.

Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1 H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein.

Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, and non-proteinaceous reporters like glutathione that may not always approximate the reactivity of protein side-chains. By contrast, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) is an industry-developed protein-based [ 1 H- 13 C]-heteronuclear multiple quantum coherence (HMQC) NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation. This Current Protocol is a users-guide to the production of the 13 C-labeled La antigen reporter protein, the reaction of test compounds with this reporter protein, as well as the collection and analysis of characteristic NMR spectra. Combined with other assay interference counter-screens, this assay will enhance chemical biology by helping researchers better prioritize chemical matter and which will increase the number of tractable HTS screening actives and aid in the development of better chemical probes., Competing Interests: Conflict of Interest Statement: The authors declare that they have no conflicts of interest.

A causal relationship between target activity modulation by small molecules and phenotypic consequence is the cornerstone of chemical biology and drug discovery. Here we articulate elements of translational chemical biology, as guideposts to ensure the appropriate use of chemical probes and the conclusions drawn from cellular studies with these molecules., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.