Selective Labeling Of Proteins Research Articles

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

AbstractQuantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N‐terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins.

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein's behavior in situ. Some proteins can be analyzed by fusion to a protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here, we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in cells. Labeling is mediated by the formation of a high-affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific for one- or two-color imaging. Last, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin) in fixed cells, highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

Photocatalytic conversion of aryl diazonium salts to sulfonyl fluorides

Sulfonyl fluorides have emerged as powerful tools in chemical biology for the selective labelling of proteins. A photocatalytic method is described for the conversion of aryl diazonium salts to aryl sulfonyl fluorides. The diazonium substrates are easily obtained in one step from functionalized anilines. We present the optimization of this mild method for the synthesis of sulfonyl fluorides, the scope of the transformation with a series of functionalized diazonium salts, and we discuss photophysical measurements that provide detailed information about the mechanism of the photochemical process.

Endogenous Cell-Surface Receptor Modification by Metal Chelation-Assisted Pyridinium Oxime Catalyst.

Organocatalyst-mediated acyl transfer reactions hold promise for selective protein labeling in biological milieu. However, they often suffer from off-target reactions and high background signals because of the requirement of high concentrations of substrates. Here, we report a new catalytic protein acylation strategy promoted by the His-tag/NiNTA interaction. The recognition-assisted activation mechanism allows efficient protein labeling even with >10-fold lower substrate concentrations than conventional reactions, thereby enabling highly selective and efficient cell-surface receptor modification in live cells.

Switching Type I/Type II Reactions by Turning a Photoredox Catalyst into a Photo-Driven Artificial Metalloenzyme

While photocatalysts have been recognized as powerful and environmentally friendly catalysts, harnessing their reactivity still remains challenging. On account of the distinctive reaction compartment that their protein scaffolds provide for the incorporated metal complex, artificial metalloenzymes (ArMs) can improve catalytic reaction rates and stereochemical selectivity. In the present study, we have developed a photo-driven ArM by incorporating a DNA photo-switch metal complex, [Ru(bpy)2dppz]2+ (1), into an apo-form riboflavin-binding protein (RFBP). We report that two potentially competing photocatalytic reaction pathways, i.e., a photoredox reaction and an energy transfer reaction, can be switched by 1 alone and by the ArM. This reaction switching was exploited in selective protein labeling; 1 alone preferentially promotes tyrosine modification, while the ArM promotes histidine modification. The present study thus opens the door for the potential use of ArMs to control the reactivity of photocatalysts.

A fluorogenic probe for SNAP-tag protein based on ESPT ratiometric signals

Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology, but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells. In this paper, we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins. In this probe, the O6-benzylguanine derivative of 3‑hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein. The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity. The excited state proton transfer occurred after illumination, to obtain the ratio fluorescence signal from blue emission to red emission, realizing the wash-free fluorescence imaging of the target proteins.

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing.

Fluorescent labels and biosensors play central roles in biological and medical research. Targeted to specific biomolecules or cells, they allow noninvasive imaging of the machinery that govern cells and organisms in real time. Recently, chemogenetic reporters made of organic dyes specifically anchored to genetic tags have challenged the paradigm of fully genetically encoded fluorescent proteins. Combining the advantage of synthetic fluorophores with the targeting selectivity of genetically encoded tags, these chemogenetic reporters open new exciting prospects for studying cell biochemistry and biology. In this Account, we present the growing toolbox of fluorescence-activating and absorption-shifting tags (FASTs), small monomeric proteins of 14 kDa (125 amino acids residues) that can be used as markers to monitor gene expression and protein localization in live cells and organisms. Engineered by directed protein evolution from the photoactive yellow protein (PYP) from the bacterium Halorhodospira halophila, prototypical FAST binds and stabilizes the fluorescent state of live-cell compatible hydroxybenzylidene rhodanine chromophores. This class of chromophores are normally dark when free in solution or in cells because they dissipate light energy through nonradiative processes. The protein cavity of FAST allows the stabilization of the deprotonated state of the chromophore and blocks the chromophore into a planar conformation, which leads to highly fluorescent protein-chromophore assemblies. The use of such fluorogenic dyes (also called fluorogens) enables the imaging of FAST fusion proteins in cells with high contrast without the need to remove unbound ligands through separate washing steps. Fluorogens with various spectral properties exist nowadays allowing investigators to adjust the spectral properties of FAST to their experimental conditions. Molecular engineering allowed furthermore to generate membrane-impermeant fluorogens for the selective labeling of cell-surface proteins. Over the years, we generated a collection of FAST variants with expanded spectral properties or fluorogen selectivity using a concerted strategy involving molecular engineering and directed protein evolution. Moreover, protein engineering allowed us to adapt FASTs for the design of fluorescent biosensors. Circular permutation enabled the generation of FAST variants with increased conformational flexibility for the design of biosensors in which fluorogen binding is conditioned to the recognition of a given analyte. Bisection of FASTs into two complementary fragments allowed us furthermore to create split variants with reversible complementation that allow the detection and imaging of dynamic protein-protein interactions. We provide, here, a general overview of the current state of development of these different systems and their applications for advanced live cell imaging and biosensing and discuss potential future directions.

Light-Controlled Traceless Protein Labeling via Decaging Thio-o-naphthoquinone Methide Chemistry.

We report the molecular design of a novel multifunctional reagent and its application for light-controlled selective protein labeling. This molecule integrates functions of protein-ligand recognition, bioconjugation, ligand cleavage, and photoactivation by merging the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand and fluorescein. Highly electrophilic o-naphthoquinone methide was photochemically released and underwent proximity-driven selective labeling with the protein of interest (e.g., carbonic anhydrases), which retains its native function after labeling.

Cyclopentadiene as a Multifunctional Reagent for Normal- and Inverse-Electron Demand Diels-Alder Bioconjugation.

Optimizing the Diels-Alder (DA) reaction for aqueous coupling has resulted in practical methods to link molecules such as drugs and diagnostic agents to proteins. Both normal electron demand (NED) and inverse electron demand (IED) DA coupling schemes have been employed, but neither mechanism entails a common multipurpose reactive group. This report focuses on expanding the bioconjugation toolbox for cyclopentadiene through the identification of reactive groups that couple through NED or IED mechanisms in aqueous solution. Dienophiles and tetrazine derivatives were screened for reactivity and selectivity toward antibodies bearing cyclopentadiene amino acids to yield bioconjugates. Twelve NED dienophiles and four tetrazine-based IED substrates were identified as capable of practical biocoupling. Furthermore, tetrazine ligation to cyclopentadiene occurred at a rate of 3.3 ± 0.5 M-1 s-1 and was capable of bioorthogonal transformations, as evidenced by the selective protein labeling in serum. Finally, an antibody-drug conjugate (ADC)-bearing monomethyl auristatin E was prepared via tetrazine conjugation to cyclopentadiene. The resulting ADC was stable and demonstrated potent activity in vitro. These findings expand the utility of cyclopentadiene as a tool to couple entities to proteins via dual DA addition mechanisms.

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane

SummaryLocalization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.

Synthetic membraneless organelles enable highly selective protein labeling for time-resolved live-cell imaging

Genetic code expansion (GCE), combined with click chemistry, offers a powerful tool for protein labeling with single-residue precision. This approach is widely suitable for visualizing proteins in both fixed and live cells, and readily compatible with advanced imaging techniques, e.g., superresolution microscopy and fluorescence-lifetime imaging microscopy (FLIM). GCE is achieved by introducing an orthogonal tRNA/synthetase suppressor pair into the living host, to reassign a stop codon to incorporate a noncanonical amino acid (ncAA).

Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry.

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal "snapshots" of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination, 3D SMLM, and protein labeling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analyzing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely, or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.

New Phenol Esters for Efficient pH-Controlled Amine Acylation of Peptides, Proteins, and Sepharose Beads in Aqueous Media.

This paper describes the discovery, synthesis, and use of novel water-soluble acylation reagents for efficient and selective modification, cross-linking, and labeling of proteins and peptides, as well as for their use in the effective modification of sepharose beads under pH control in aqueous media. The reagents are based on a 2,4-dichloro-6-sulfonic acid phenol ester core combined with a variety of linker structures. The combination of these motifs leads to an ideal balance between hydrolytic stability and reactivity. At high pH, good to excellent conversions (up to 95%) and regioselectivity (up to 99:1 Nε/Nα amine ratio) in the acylation were realized, exemplified by the chemical modification of incretin peptides and insulin. At neutral pH, an unusually high preference toward the N-terminal phenylalanine in an insulin derivative was observed (>99:1 Nα/Nε), which is up until now unprecedented in the literature for more elaborate reagents. In addition, the unusually high hydrolytic stability of these reagents and their ability to efficiently react at low concentrations (28 μM or 0.1 mg/mL) are exemplified with a hydroxy linker-based reagent and are a unique feature of this work.

Exploring Ligand-Directed N-Acyl-N-alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development.

Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting N-acyl-N-alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4CRBN E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question.

Aptamer C10.36 Reveals a Ribonucleoprotein Complex on the Surface of Non-Hodgkin Lymphoma Cells Providing Candidates for Multi-Target Therapeutics

Introduction: Aptamers are valuable tools for identifying novel therapeutic targets due to their high affinity and specificity. The relative ease of selection of aptamers binding to complex targets and their lack of immunogenicity have led to the incorporation of aptamers into drug transport vehicles and cell labeling tools. However, their potential as direct anti-cancer therapies remains to be explored. Recent advances in aptamer therapeutics have underscored their potential to meet the need for better therapies in B-cell non-Hodgkin lymphomas (NHL). For example, the aptamer AS1411 binds nucleolin and inhibits the proliferation of multiple leukemia and lymphoma cancer cell lines. The DNA aptamer C10.36 has been shown to selectively bind to Ramos Burkitt lymphoma cells and to be internalized via clathrin-dependent endocytosis. Here we identify the molecular target of C10.36 and explore its potential role as a targeted therapy in NHL.Methods: The protein targets of C10.36 on the surface of tumor cells were identified by LC-MS/MS following affinity purification using biotin labeled aptamers. Direct molecular targets of C10.36 were identified by aptamer-based affinity labeling of target proteins after photo-crosslinking and subsequent affinity purification. Bands were excised from polyacrylamide gels, reduced, alkylated with an Iodo-TMT126/129 label, digested, and analyzed by LC-MS/MS. Targets were confirmed by the presence of the precursor mass of the peptide with the TMT-SDAD label and both the 126 and 129 reporter ions in the MS/MS spectrum. To confirm extracellular expression of proteins on intact cells, we carried out selective biotin labeling of surface proteins followed by subsequent purification and LC-MS/MS detection. In vitro anti-proliferative activity of C10.36 was assessed by CellTiter-Glo® assay. Preferential binding and internalization of C10.36 were assessed by flow cytometry and immunofluorescence, respectively.Results: C10.36 affinity pull-downs revealed that it associates with a ribonucleoprotein complex on the surface of Ramos cells. Gene Ontology enrichment analysis revealed proteins over-represented in chromatin organization and RNA metabolic process gene sets. Covalent crosslinking identified heterogeneous nuclear ribonucleoprotein U (hnRNP U) as a direct binding target of C10.36 within the complex. We next surveyed additional lymphoma and leukemia cell lines for C10.36 binding. Interestingly, C10.36 binding to MEC-1 and SU-DHL-1 cells was readily observed, whereas binding to a normal B cell derivative, SKW6.4, was greatly reduced. Although hnRNP U was detected on the surface of all four cell lines by cell surface protein labeling, affinity purification of hnRNP U by C10.36 was only observed in the fully transformed cell lines and not in SKW6.4 cells. This observation indicates that C10.36 binding may be determined by conformation, context or post-translational modification. Treatment with C10.36 led to a loss of viability and IC50 values for sensitive cell lines ranged from 100 to 400 nM. Importantly, a single point mutation in C10.36 (G24A) abrogated both binding and anti-proliferative activity.Conclusion: The present study identifies cell surface hnRNP U and its ribonucleoprotein interaction partners as potential therapeutic targets for NHL and highlights the potential for the development of C10.36 as a novel anti-B cell lymphoma targeted therapy. DisclosuresTonapi:Caris Life Sciences: Employment. Duncan:Caris Life Sciences: Employment. Vaishali:Caris Life Sciences: Employment. Rosenow:Caris Life Sciences: Employment. Richards:Caris Life Sciences: Employment. Tinder:Caris Life Sciences: Employment. O'Neill:Caris Life Sciences: Employment. Miglarese:Caris Life Sciences: Employment. Spetzler:Caris Life Sciences: Employment. Famulok:Caris Life Sciences: Consultancy. Mayer:Caris Life Sciences: Consultancy.

Selective Mitochondrial Protein Labeling Enabled by Biocompatible Photocatalytic Reactions inside Live Cells.

Biocompatible reactions are powerful tools to probe protein functions in their native environment. Due to the difficulty of penetrating the live-cell membrane and the complex intracellular environment, the biocompatible reactions inside live cells are challenging, especially at the subcellular level with spatial resolution. Here we report the first biocompatible photocatalytic azide conjugation reaction inside live cells to achieve the mitochondria-selective proteins labeling. The organic dyes acridine orange, fluorescein, and rhodamine 123 were developed as the biocompatible photocatalysts for the proteins labeling with aryl azides, which yielded benzazirines and ketenimines from triplet nitrenes for the protein nucleophilic residue trapping. The photocatalytic azide conjugation reaction with rhodamine 123 selectively labeled the mitochondrial proteins via the organic dye’s mitochondrial localization. In response to the mitochondrial stress induced by rotenone, this photocatalytic azide-promoted labeling method mapped the dynamic mitochondrial proteome changes with high temporal-spatial precision and identified several potential mitochondrial stress-response proteins for the first time. The high temporal-spatial precision of this photocatalytic azide-promoted labeling method holds excellent potential for intracellular protein network investigations.

Fluorogen‐Activating Proteins as a powerful new imaging tool for quantitative protein trafficking studies in yeast

Recent advantages of genetically encoded fluorescent probes have led to the development of fluorogen-activating proteins (FAPs). This technology has two components: a non-fluorescent single chain antibody (SCA) that can be fused to a protein of interest and fluorogens, which in our case are derivatives of malachite green that are non-fluorescent when free in solution. When the SCA and fluorogen bind, there is a 20,000-fold fluorescent increase relative to unbound dye. The FAP-technology has two major advantages to standard fluorescent probes: i) the fluorogen exists as either a membrane-permeant or impermeant form and this allows for selective labeling of cell surface proteins and (2) since SCA does not fluoresce when it is not bound by dye and so there is no background fluorescence during the imaging of other markers until after the fluorogen is added. Although developed in yeast, this technology surprisingly has not been applied in this model system until our recent studies. In order to make this technology more available to the yeast community, we have optimized the SCA sequence for yeast expression, created a series for FAP-tagged plasmid constructs, and generated a suite of intracellular localization markers to aid in cell biology studies in yeast. We further employ the FAP technology to study the trafficking of the G-protein coupled receptor Ste3 and its regulation by the alpha-arrestins, an important class of trafficking adaptor.