RESUMEN
Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.
Asunto(s)
Péptidos , Fluorescencia , Cinética , Preparaciones Farmacéuticas , TermodinámicaRESUMEN
6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.
Asunto(s)
Cartilla de ADN/química , Nucleótidos de Desoxiuracil/química , Desoxiuridina/análogos & derivados , Triazinas/química , Tubercidina/análogos & derivados , Reacción de Cicloadición , Cartilla de ADN/síntesis química , ADN Polimerasa Dirigida por ADN/química , Nucleótidos de Desoxiuracil/síntesis química , Reacción en Cadena de la Polimerasa , Triazinas/síntesis química , Tubercidina/síntesis química , Tubercidina/químicaRESUMEN
The proteolytic complex ClpXP is fundamental to bacterial homeostasis and pathogenesis. Because of its conformational flexibility, the development of potent ClpXP inhibitors is challenging, and novel tools to decipher its intricate regulation are urgently needed. Herein, we present amino acid based phenyl esters as molecular probes to study the activity and oligomerization of the ClpXP complex of S.â aureus. Systematic screening of (R)- and (S)-amino acids led to compounds showing potent inhibition, as well as stimulation of ClpXP-mediated proteolysis. Substoichiometric binding of probes arrested ClpXP in an unprecedented heptamer-hexamer assembly, in which the two heptameric ClpP rings are dissociated from each other. At the same time, the affinity between ClpX and ClpP increased, leading to inhibition of both enzymes. This conformational arrest is beneficial for the consolidated shutdown of ClpXP, as well as for the study of the oligomeric state during its catalytic cycle.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Endopeptidasa Clp/antagonistas & inhibidores , Ésteres/farmacología , Multimerización de Proteína/efectos de los fármacos , Proteolisis/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Staphylococcus aureus/enzimología , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/metabolismo , Ésteres/química , Péptidos/química , Péptidos/farmacología , Conformación Proteica , Inhibidores de Serina Proteinasa/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The characterization of novel therapeutic antibodies with multivalent or multispecific binding sites requires new measurement modalities for biosensors, to discriminate the engagement of antigens via one, two, or even more binding moieties. The presentation of antigens on a sensor surface in a well-controlled spatial arrangement is a prerequisite for the successful interpretation of binding kinetics measurements of multivalent analytes, but the adjustment of defined distances between immobilized ligands is difficult to achieve in state-of-the-art biosensor systems. Here, we introduce a simple DNA nanostructure resembling a slingshot, which can be configured with two identical or two different antigens (bivalent or bispecific), which are spaced at a defined distance. We characterize the slingshot structure with a chip-based biosensor using electrically switchable DNA nanolevers and demonstrate that bivalent and monovalent antibodies selectively interact with slingshots that have been functionalized with two identical or two different antigens, respectively. The dissociation kinetics are quantified in real-time measurements and we show that the slingshot structure enables a clear differentiation between affinity and avidity effects.
Asunto(s)
Anticuerpos/análisis , ADN/química , Nanoestructuras/química , Anticuerpos/inmunología , Antígenos/química , Antígenos/inmunología , Técnicas Biosensibles/métodos , ADN/genética , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Termodinámica , Temperatura de TransiciónRESUMEN
A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1-methylcyclopropene (1MCP) group at the 7-position of 7-deaza-2'-deoxyadenosine and applied for primer extension experiments. The real-time kinetic data reveals that this 1MCP-modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP-modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site-specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.
Asunto(s)
Ciclopropanos/química , Cartilla de ADN/química , ADN/análisis , Desoxiadenosinas/química , Colorantes Fluorescentes/química , Secuencia de Bases , Células HeLa , Humanos , Metilación , Microscopía Fluorescente , Imagen ÓpticaRESUMEN
The strawberry fruit allergens Fra a 1.01E, Fra a 1.02 and Fra a 1.03 belong to the group of pathogenesis-related 10 (PR-10) proteins and are homologs of the major birch pollen Bet v 1 and apple allergen Mal d 1. Bet v 1 related proteins are the most extensively studied allergens but their physiological function in planta remains elusive. Since Mal d 1-Associated Protein has been previously identified as interaction partner of Mal d 1 we studied the binding of the orthologous Fra a 1-Associated Protein (FaAP) to Fra a 1.01E/1.02/1.03. As the C-terminal sequence of FaAP showed strong auto-activation activity in yeast 2-hybrid analysis a novel time resolved DNA-switching system was successfully applied. Fra a 1.01E, Fra a 1.02, and Fra a 1.03 bind to FaAP with KD of 4.5 ± 1.1, 15 ± 3, and 11 ± 2 nM, respectively. Fra a 1.01E forms a dimer, whereas Fra a 1.02 and Fra a 1.03 bind as monomer. The results imply that PR-10 proteins might be integrated into a protein-interaction network and FaAP binding appears to be essential for the physiological function of the Fra a 1 proteins.
Asunto(s)
Antígenos de Plantas/química , Fragaria/química , Proteínas de Plantas/química , Secuencia de Aminoácidos/genética , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Humanos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas/genéticaRESUMEN
The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Técnicas Biosensibles , Inmunoprecipitación de Cromatina , Técnicas Electroquímicas , Cinética , Microelectrodos , Unión Proteica , TermodinámicaRESUMEN
Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a ß-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.
Asunto(s)
Daño del ADN , Enzimas Reparadoras del ADN/química , Reparación del ADN , Proteínas de Saccharomyces cerevisiae/química , 2-Acetilaminofluoreno/química , 2-Acetilaminofluoreno/metabolismo , Secuencia de Aminoácidos , Cisplatino/química , Cisplatino/metabolismo , Cristalografía por Rayos X , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Termodinámica , Temperatura de TransiciónRESUMEN
The toxic nature of polycyclic aromatic hydrocarbons (PAHs), in particular benzo[a]pyrene (B[a]P), neccessitates the monitoring of PAH contamination levels in food and the environment. Here we introduce an indirect immunoassay format using electro-switchable biosurfaces (ESB) for the detection of B[a]P in water. The association of anti-B[a]P antibodies to microelectrodes is analyzed in real-time by measuring changes in the oscillation dynamics of DNA nanolever probes, which are driven to switch their orientations by high-frequency electrical actuation. From the association kinetics, the active concentration of anti-B[a]P, and hence the B[a]P contamination of the sample, can be determined with picomolar sensitivity. The detection limit of the assay improves with measurement time because increasingly accurate analyses of the binding kinetics become possible. It is demonstrated that an exceedance of the permissible 10 ng/L (40 pM) limit for B[a]P is detectable in an unprecedented short assay time (<1 h), using a simple three-step workflow involving minimal sample preparation. The reproducibility was satisfying with standard deviations below 5%. Further, the utility of the assay for practical applications is exemplified by analyzing a river water sample.
Asunto(s)
Benzo(a)pireno/análisis , Carcinógenos/análisis , Contaminantes Químicos del Agua/análisis , Anticuerpos/inmunología , Benzo(a)pireno/química , Carcinógenos/química , Sondas de ADN/análisis , Sondas de ADN/metabolismo , Electrones , Inmunoensayo , Cinética , Microelectrodos , Ríos/química , Propiedades de Superficie , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/inmunologíaRESUMEN
Living organisms protect the genome against external influences by recognizing and repairing damaged DNA. A common source of gene mutation is the oxidized guanine, which undergoes base excision repair through cleavage of the glycosidic bond between the ribose and the nucleobase of the lesion. We unravel the repair mechanism utilized by bacterial glycosylase, MutM, using quantum-chemical calculations involving more than 1000â atoms of the catalytic site. In contrast to the base-protonated pathway currently favored in the literature, we show that the initial protonation of the lesion's ribose paves the way for an almost barrier-free glycosidic cleavage. The combination of theoretical and experimental data provides further insight into the selectivity and discrimination of MutM's binding site toward various substrates.
Asunto(s)
Reparación del ADN , ADN/química , ADN/metabolismo , Protones , Ribosa/química , ADN Glicosilasas/metabolismo , Modelos Moleculares , Estructura Molecular , Ribosa/metabolismoRESUMEN
Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements.
Asunto(s)
Proteínas Bacterianas/análisis , Gonadotropina Coriónica/análisis , ADN/química , Proteínas Fúngicas/análisis , Inmunoglobulina G/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Gonadotropina Coriónica/química , Gonadotropina Coriónica/metabolismo , Electricidad , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Cinética , Simulación de Dinámica Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of ß2-glycoprotein I (ß2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of ß2-GPI to the cardiolipin surface. Using the switchSENSE sensor, ß2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between ß2-GPI-dependent autoimmune- and ß2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.
Asunto(s)
Anticuerpos Anticardiolipina/inmunología , Técnicas Biosensibles , Cardiolipinas/química , Oro/química , Anticuerpos Anticardiolipina/química , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/inmunología , ADN/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Cinética , Ligandos , Microscopía de Túnel de Rastreo/métodos , Modelos Químicos , Conformación Molecular , Resonancia por Plasmón de Superficie/métodos , Propiedades de Superficie , Factores de Tiempo , beta 2 Glicoproteína I/químicaRESUMEN
A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers' dynamic motion. We demonstrate the quantitation of binding kinetics (k(on), k(off) rate constants), dissociation constants (K(D) in the pM regime), and the influence of competitive binders (EC(50) values). Moreover, the "switchSENSE" method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA(3)) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins' individual chemical environments significantly influence the His(6)-NTA(3) interaction.
Asunto(s)
Proteínas/química , Cinética , Ligandos , Unión Proteica , Propiedades de SuperficieAsunto(s)
Sondas de ADN/química , Proteínas de Unión al ADN/análisis , Desoxiguanosina/análogos & derivados , Proteoma/química , 8-Hidroxi-2'-Desoxicoguanosina , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/química , Sondas de ADN/síntesis química , Reparación del ADN , Proteínas de Unión al ADN/química , Bases de Datos de Proteínas , Desoxiguanosina/química , Diazometano/química , Humanos , Procesos Fotoquímicos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
An investigation of the precise interactions between damaged DNA and DNA repair enzymes is required in order to understand the lesion recognition step, which is one of the most fundamental processes in DNA repair. Most recently, photoaffinity labeling approaches have enabled the analysis of even transient protein-DNA interactions. Here we report the synthesis and evaluation of oligonucleotides that contain two photoaffinity "catcher moieties" next to incorporated DNA lesions. With these DNA constructs it is possible to analyze the interactions between DNA lesions and the appropriate repair enzymes. The probes labeled the repair protein efficiently enough to enable subsequent protein analysis by mass spectrometry.