RESUMO
Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.
Assuntos
DNA/química , Fragmentos de Imunoglobulinas/química , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/química , Alcinos/química , Azidas/química , Catálise , Linhagem Celular , Cobre/química , Reação de Cicloadição , Proteínas de Fluorescência Verde/química , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Anticorpos de Domínio Único/químicaRESUMO
Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.
Assuntos
Separação Celular/métodos , Antígenos de Histocompatibilidade , Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologia , Animais , Antígenos de Histocompatibilidade/análise , Humanos , Receptores de Antígenos de Linfócitos T/análiseRESUMO
Herein, the application of N-hydroxysuccinimide-modified phosphonamidate building blocks for the incorporation of cysteine-selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo-crosslinking side products that can occur with commonly applied succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody-drug conjugates, analogously to FDA-approved, SMCC-linked Kadcyla, and to the synthesis of a functional antibody-protein conjugate.
Assuntos
Amidas/química , Etilenoglicol/química , Proteínas de Fluorescência Verde/química , Ácidos Fosfóricos/química , Succinimidas/química , Linhagem Celular Tumoral , Humanos , Estrutura MolecularRESUMO
We introduce a chemoenzymatic strategy for straightforward in vitro generation of C-terminally linked fusion proteins. Tubulin tyrosine ligase is used for the incorporation of complementary click chemistry handles facilitating subsequent formation of functional bispecific antibody-fragments. This simple strategy may serve as central conjugation hub for a modular protein ligation platform.
Assuntos
Anticorpos/química , Peptídeo Sintases/química , Proteínas Recombinantes de Fusão/química , Anticorpos/metabolismo , Química Click , Estrutura Molecular , Peptídeo Sintases/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Requirements for novel bioconjugation reactions for the synthesis of antibody-drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate-linked ADCs have excellent properties for next-generation antibody therapeutics in terms of serum stability and inâ vivo antitumor activity.
Assuntos
Antineoplásicos Imunológicos/química , Cisteína/química , Etilenoglicol/química , Imunoconjugados/metabolismo , Organofosfonatos/química , Receptor ErbB-2/imunologia , Trastuzumab/química , Antineoplásicos Imunológicos/imunologia , Humanos , Imunoconjugados/química , Trastuzumab/imunologia , Células Tumorais CultivadasRESUMO
We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.
Assuntos
Antineoplásicos Imunológicos/química , Cisteína/química , Imunoconjugados/metabolismo , Organofosfonatos/química , Receptor ErbB-2/imunologia , Compostos de Sulfidrila/química , Trastuzumab/química , Antineoplásicos Imunológicos/metabolismo , Cisteína/metabolismo , Humanos , Imunoconjugados/química , Iodoacetamida/química , Iodoacetamida/metabolismo , Maleimidas/química , Maleimidas/metabolismo , Organofosfonatos/metabolismo , Compostos de Sulfidrila/metabolismo , Trastuzumab/metabolismoRESUMO
Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.
Assuntos
Neoplasias/imunologia , Anticorpos de Domínio Único/química , Aminoácidos/química , Aminoácidos/imunologia , Animais , Antígenos/química , Antígenos/imunologia , Humanos , Espectrometria de Massas , Neoplasias/patologia , Anticorpos de Domínio Único/imunologiaRESUMO
PURPOSE: Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. METHODS: Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. RESULTS: The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. CONCLUSION: Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.
Assuntos
Nanopartículas/administração & dosagem , Proteínas/administração & dosagem , Anticorpos de Domínio Único/administração & dosagem , Linhagem Celular , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Citoplasma/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Células KB , Nanopartículas/metabolismo , Transporte Proteico , Proteínas/metabolismo , Anticorpos de Domínio Único/metabolismoRESUMO
Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals, combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability.
Assuntos
Biotecnologia , Imunoconjugados , Aminoácidos/química , Animais , Biotecnologia/métodos , Biotecnologia/tendências , Dissulfetos/química , Humanos , Imunoconjugados/química , Imunoconjugados/genética , Terapia de Alvo Molecular/tendênciasRESUMO
A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin.
Assuntos
Peptídeo Sintases/metabolismo , Tirosina/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Peptídeo Sintases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tirosina/químicaRESUMO
Topoisomerase I (TOP1) Inhibitors constitute an emerging payload class to engineer antibody-drug conjugates (ADC) as next-generation biopharmaceutical for cancer treatment. Existing ADCs are using camptothecin payloads with lower potency and suffer from limited stability in circulation. With this study, we introduce a novel camptothecin-based linker-payload platform based on the highly potent camptothecin derivative exatecan. First, we describe general challenges that arise from the hydrophobic combination of exatecan and established dipeptidyl p-aminobenzyl-carbamate (PAB) cleavage sites such as reduced antibody conjugation yields and ADC aggregation. After evaluating several linker-payload structures, we identified ethynyl-phosphonamidates in combination with a discrete PEG24 chain to compensate for the hydrophobic PAB-exatecan moiety. Furthermore, we demonstrate that the identified linker-payload structure enables the construction of highly loaded DAR8 ADCs with excellent solubility properties. Head-to-head comparison with Enhertu, an approved camptothecin-based ADC, revealed improved target-mediated killing of tumor cells, excellent bystander killing, drastically improved linker stability in vitro and in vivo and superior in vivo efficacy over four tested dose levels in a xenograft model. Moreover, we show that ADCs based on the novel exatecan linker-payload platform exhibit antibody-like pharmacokinetic properties, even when the ADCs are highly loaded with eight drug molecules per antibody. This ADC platform constitutes a new and general solution to deliver TOP1 inhibitors with highest efficiency to the site of the tumor, independent of the antibody and its target, and is thereby broadly applicable to various cancer indications.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Anticorpos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/químicaRESUMO
The recent success of antibody-drug conjugates (ADC), exemplified by seven new FDA-approvals within three years, has led to increased attention for antibody based targeted therapeutics and fueled efforts to develop new drug-linker technologies for improved next generation ADCs. We present a highly efficient phosphonamidate-based conjugation handle that combines a discrete hydrophilic PEG-substituent, an established linker-payload and a cysteine-selective electrophile in one compact building block. This reactive entity provides homogeneous ADCs with a high drug-to-antibody ratio (DAR) of 8 in a one-pot reduction and alkylation protocol from non-engineered antibodies. The compact branched PEG-architecture introduces hydrophilicity without increasing the distance between antibody and payload, allowing the generation of the first homogeneous DAR 8 ADC from VC-PAB-MMAE without increased in vivo clearance rates. This high DAR ADC exhibits excellent in vivo stability and increased antitumor activity in tumour xenograft models relative to the established FDA approved VC-PAB-MMAE ADC Adcetris, clearly showing the benefit of the phosphonamidate based building-blocks as a general tool for the efficient and stable antibody-based delivery of highly hydrophobic linker-payload systems.
RESUMO
Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.
Assuntos
Camelídeos Americanos/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Nanopartículas de Magnetita , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Animais , Camelídeos Americanos/genética , Fragmentos de Imunoglobulinas/genética , Imunoprecipitação , Proteínas Luminescentes/metabolismo , Magnetospirillum/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Vermelha FluorescenteRESUMO
The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, distribution, modification, and interaction of all cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not about endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications, and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (V(H)H) from Camelidae. These extremely stable V(H)H domains can be produced in bacteria, coupled to matrices, and used for affinity purification and proteome studies. Alternatively, these V(H)H domains can be fused with fluorescent proteins and expressed in living cells. These fluorescent antigen-binding proteins called "chromobodies" can be used to detect and trace proteins and other cellular components in vivo. Chromobodies can, in principle, detect any antigenic structure, including posttranslational modifications, and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analysis. Depending on the epitope chosen, chromobodies can also be used to modulate protein function in living cells.
Assuntos
Anticorpos/química , Imunofluorescência/métodos , Proteínas/química , Proteoma/química , Animais , Anticorpos/genética , Anticorpos/metabolismo , Linhagem Celular , Células/química , Células/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Conformação Proteica , Proteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Tub-tag labeling, a novel chemoenzymatic protein functionalization method, facilitates one-step fluorescent labeling of functional biomolecules. The enzyme tubulin tyrosine ligase incorporates coumarin-amino acids to the terminal carboxylic acid of proteins containing a short peptidic recognition sequence called Tub-tag. Here we describe the one-step Tub-tag protein modification protocol in detail and explain its utilization to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics.
Assuntos
Peptídeo Sintases/química , Peptídeos/química , Proteínas/isolamento & purificação , Coloração e Rotulagem/métodos , Aminoácidos/química , Corantes Fluorescentes/química , Proteínas/químicaRESUMO
Tubulin tyrosine ligase (TTL) catalyzes the addition of tyrosine derivatives to the C-terminal carboxylic acid of proteins. The enzyme binds to a 14-amino acid recognition sequence, termed Tub-tag, and allows for the introduction of tyrosine derivatives that carry a unique chemical handle. These handles enable subsequent bioorthogonal reactions with a great variety of probes or effector molecules. Clearly, this two-step chemoenzymatic approach, facilitates the site-specific functionalization of proteins. Furthermore, due to its broad substrate tolerance, tubulin tyrosine ligase also enables an enzymatic one-step modification. For example, a coumarin amino acid was utilized to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics. Here we describe the modification of proteins using TTL in detail via a one-step as well as two-step procedure and highlight its practicability for applications in imaging, diagnostics, and cell biology.
Assuntos
Peptídeo Sintases/metabolismo , Proteínas/metabolismo , Aminoácidos/química , Catálise , Linhagem Celular , Humanos , Peptídeo Sintases/química , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-AtividadeRESUMO
Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics.
Assuntos
Aminoácidos/química , Proteínas/química , Coloração e Rotulagem , Aminoácidos/metabolismo , Biotinilação , Química Click , Clonagem Molecular , Expressão Gênica , Humanos , Modelos Moleculares , Estrutura Molecular , Peptídeo Sintases/metabolismo , Conformação Proteica , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteólise , Coloração e Rotulagem/métodos , Tirosina/metabolismoRESUMO
Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods.
Assuntos
Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia , Núcleo Celular/química , Células HeLa , Humanos , Tamanho da Partícula , Porosidade , Proteínas/química , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/genética , Pontos de Checagem do Ciclo Celular/genética , Ensaios de Triagem em Larga Escala , Receptor ErbB-2/genética , Trastuzumab/farmacologia , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Feminino , Expressão Gênica , Humanos , Indóis/química , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Sensibilidade e EspecificidadeRESUMO
The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.