RESUMEN
BACKGROUND: Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. RESULTS: We developed an efficient approach to purify functionalized protein nanostructures directly from bacterial lysates through a type of multimodal chromatography (MMC) that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. We employed the ultrastable filamentous protein gamma-prefoldin as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using MMC was tested across a wide range of salt concentrations and pH, demonstrating that the MMC resin is robust, however the optimal choice of salt species, salt concentration, and pH is likely dependent on the protein nanostructure to be purified. In addition, we show that pre-processing of the samples with tangential flow filtration to remove nucleotides and metabolites improves resin capacity, and that post-processing with Triton X-114 phase partitioning is useful to remove lipids and any remaining lipid-associated protein. Subsequently, functionalized protein filaments were purified from bacterial lysates using MMC and shown to be free of unincorporated subunits. The assembly and purification of protein filaments with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Förster resonance energy transfer, and transmission electron microscopy. Finally, we compared our MMC workflow to anion exchange chromatography with the purification of encapsulin nanocompartments containing a fluorescent protein as a cargo, demonstrating the versatility of the protocol and that the purity of the assembly is comparable to more traditional procedures. CONCLUSIONS: We envision that the use of MMC will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.
Asunto(s)
Cromatografía , Nanoestructuras , Cromatografía/métodos , Proteínas Recombinantes , Nanoestructuras/química , Materiales Biocompatibles , Proteínas BacterianasRESUMEN
The structural diversity of natural products offers unique opportunities for drug discovery, but challenges associated with their isolation and screening can hinder the identification of drug-like molecules from complex natural product extracts. Here we introduce a mass spectrometry-based approach that integrates untargeted metabolomics with multistage, high-resolution native mass spectrometry to rapidly identify natural products that bind to therapeutically relevant protein targets. By directly screening crude natural product extracts containing thousands of drug-like small molecules using a single, rapid measurement, we could identify novel natural product ligands of human drug targets without fractionation. This method should significantly increase the efficiency of target-based natural product drug discovery workflows.
Asunto(s)
Productos Biológicos/química , Ligandos , Proteínas/química , Productos Biológicos/metabolismo , Anhidrasa Carbónica I/química , Anhidrasa Carbónica I/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Metabolómica/métodos , Proteínas/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been primarily used for the discrimination of bacterial strains. Reproducible DNA fingerprint patterns for various phage types were generated using either of the two primers. RESULTS: The similarity index of replicates ranged from 89.4-100% for BOXA2R-PCR, and from 90 to 100% for BOXA1R-PCR. The method of DNA isolation (p = 0.08) and the phage propagation conditions at two different temperatures (p = 0.527) had no significant influence on generated patterns. Rep-PCR amplification products were generated from different templates including purified phage DNA, phage lysates and phage plaques. The use of this method enabled comparisons of phage genetic profiles to establish their similarity to related or unrelated phages and their bacterial hosts. CONCLUSION: The findings suggest that repetitive-PCR could be used as a rapid and inexpensive method to preliminary screen phage isolates prior to their selection for more comprehensive studies. The adoption of this rapid, simple and reproducible technique could facilitate preliminary characterisation of a large number of phage isolates and the investigation of genetic relationship between phage genotypes.
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Bacteriófagos/clasificación , Cartilla de ADN/genética , Técnicas de Genotipaje/métodos , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , ADN Viral/genética , Filogenia , Reacción en Cadena de la Polimerasa , TemperaturaRESUMEN
Methylation of arginine residues in proteins, an enzyme-mediated post-translational modification (PTM), is important for mRNA processing and transport and for the regulation of many protein-protein interactions. However, proteolytic peptides resulting from alternative sites of post-translational methylation have identical masses and cannot be readily separated by standard liquid chromatography-mass spectrometry. Unlike acetylation or phosphorylation, methylation of arginine does not strongly affect the charge states of peptide ions, multiple instances of methylation can occur on a single amino acid residue, and the relative mass of the modification is <1% that of the typical proteolytic peptide. High field asymmetric waveform ion mobility spectrometry (FAIMS) is an orthogonal separation method to liquid chromatography that can rapidly separate gaseous ions prior to detection by mass spectrometry. Here, we report that FAIMS can be used to separate arginine-methylated peptides that differ by the position of a single methyl group for both mono- and dimethylated variants. Although the resolution of separation for these arginine-methylated peptides improved with increasing amounts of helium in the FAIMS carrier gas as expected, we found that the site of methylation can strongly affect the dependence of the electric field used for ion transmission on the extent of helium in the carrier gas. Thus, certain isobaric peptides can be cotransmitted at high helium concentrations whereas lower concentrations can be used for successful separations of such peptide mixtures. The capability to rapidly resolve isobaric arginine-methylated peptides should be useful in the future for the detailed analysis of protein arginine methylation in biological samples.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas Nucleares/química , Péptidos/aislamiento & purificación , Ribonucleoproteínas Nucleolares Pequeñas/química , Proteínas de Saccharomyces cerevisiae/química , Arginina/análogos & derivados , Arginina/química , Arginina/metabolismo , Helio/química , Espectrometría de Movilidad Iónica/métodos , Metilación , Proteínas Nucleares/metabolismo , Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias Repetitivas de Aminoácido , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys(79) in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys(79) methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.
Asunto(s)
Metiltransferasas/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Lisina/metabolismo , Espectrometría de Masas/métodos , Metilación , Metiltransferasas/genética , Mutación , Factor 1 de Elongación Peptídica/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Post-translational modifications of proteins (PTMs) act as key regulators of protein activity and of protein-protein interactions (PPIs). To date, it has been difficult to comprehensively explore functional links between PTMs and PPIs. To address this, we developed PTMOracle, a Cytoscape app for coanalyzing PTMs within PPI networks. PTMOracle also allows extensive data to be integrated and coanalyzed with PPI networks, allowing the role of domains, motifs, and disordered regions to be considered. For proteins of interest, or a whole proteome, PTMOracle can generate network visualizations to reveal complex PTM-associated relationships. This is assisted by OraclePainter for coloring proteins by modifications, OracleTools for network analytics, and OracleResults for exploring tabulated findings. To illustrate the use of PTMOracle, we investigate PTM-associated relationships and their role in PPIs in four case studies. In the yeast interactome and its rich set of PTMs, we construct and explore histone-associated and domain-domain interaction networks and show how integrative approaches can predict kinases involved in phosphodegrons. In the human interactome, a phosphotyrosine-associated network is analyzed but highlights the sparse nature of human PPI networks and lack of PTM-associated data. PTMOracle is open source and available at the Cytoscape app store: http://apps.cytoscape.org/apps/ptmoracle .
Asunto(s)
Aplicaciones Móviles , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Fúngicas , Humanos , Fosfotransferasas/metabolismo , Fosfotirosina/metabolismo , Proteínas , LevadurasRESUMEN
In recent years, protein methylation has been established as a major intracellular PTM. It has also been proposed to modulate protein-protein interactions (PPIs) in the interactome. To investigate the effect of PTMs on PPIs, we recently developed the conditional two-hybrid (C2H) system. With this, we demonstrated that arginine methylation can modulate PPIs in the yeast interactome. Here, we used the C2H system to investigate the effect of lysine methylation. Specifically, we asked whether Ctm1p-mediated trimethylation of yeast cytochrome c Cyc1p, on lysine 78, modulates its interactions with Erv1p, Ccp1p, Cyc2p and Cyc3p. We show that the interactions between Cyc1p and Erv1p, and between Cyc1p and Cyc3p, are significantly increased upon trimethylation of lysine 78. This increase of interaction helps explain the reported facilitation of Cyc1p import into the mitochondrial intermembrane space upon methylation. This first application of the C2H system to the study of methyllysine-modulated interactions further confirms its robustness and flexibility.
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Citocromos c/metabolismo , Lisina/metabolismo , Western Blotting , Escherichia coli/metabolismo , Espectrometría de Masas , Metilación , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Many proteins, including p53, the FoxO transcription factors, RNA polymerase II, pRb, and the chaperones, have extensive post-translational modifications (PTMs). Many of these modifications modulate protein-protein interactions, controlling interaction presence/absence and specificity. Here we propose the notion of the interaction code, a widespread means by which modifications are used to control interactions in the proteome. Minimal interaction codes are likely to exist on proteins that have two modifications and two or more interaction partners. By contrast, complex interaction codes are likely to be found on "date hub" proteins that have many interactions, many PTMs, or are targeted by many modifying and demodifying enzymes. Proteins with new interaction codes should be discoverable by examining protein interaction networks, annotated with PTMs and protein-modifying enzyme-substrate links. Multiple instances or combinations of phosphorylation, acetylation, methylation, O-GlcNAc, or ubiquitination will likely form interaction codes, especially when colocated on a protein's single interaction interface. A network-based example of code discovery is given, predicting the yeast protein Npl3p to have a methylation/phosphorylation-dependent interaction code.
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Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Acetilación , Animales , Sitios de Unión , Proteínas Fúngicas/metabolismo , Glicosilación , Humanos , Metilación , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Transducción de Señal , UbiquitinaciónRESUMEN
Desmin, the muscle-specific intermediate filament, is involved in myofibrillar myopathies, dilated cardiomyopathy and muscle wasting. Desmin is the target of posttranslational modifications (PTMs) such as phosphorylation, ADP-ribosylation and ubiquitylation as well as nonenzymatic modifications such as glycation, oxidation and nitration. Several PTM target residues and their corresponding modifying enzymes have been discovered in human and nonhuman desmin. The major effect of phosphorylation and ADP-ribosylation is the disassembly of desmin filaments, while ubiquitylation of desmin leads to its degradation. The regulation of the desmin filament network by phosphorylation and ADP-ribosylation was found to be implicated in several major biological processes such as myogenesis, myoblast fusion, muscle contraction, muscle atrophy, cell division and possibly desmin interactions with its binding partners. Phosphorylation of desmin is also implicated in many forms of desmin-related myopathies (desminopathies). In this review, we summarize the findings on desmin PTMs and their implication in biological processes and pathologies, and discuss the current knowledge on the regulation of the desmin network by PTMs. We conclude that the desmin filament network can be seen as an intricate scaffold for muscle cell structure and biological processes and that its dynamics can be affected by PTMs. There are now precise tools to investigate PTMs and visualize cellular structures that have been underexploited in the study of desminopathies. Future studies should focus on these aspects.
Asunto(s)
Cardiomiopatías/genética , Desmina/genética , Enfermedades Musculares/genética , Distrofias Musculares/genética , Procesamiento Proteico-Postraduccional/genética , Animales , Pollos , Cricetinae , Desmina/biosíntesis , Humanos , Filamentos Intermedios , Ratones , Células Musculares/citología , Células Musculares/metabolismo , Contracción Muscular , Músculos/citologíaRESUMEN
Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo-designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between "on" and "off" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.
Asunto(s)
Procesamiento Proteico-Postraduccional , Fosforilación , Cinética , Unión Proteica , Proteínas/metabolismo , Proteínas/química , Ingeniería de Proteínas/métodosRESUMEN
Many bacteria swim driven by an extracellular filament rotated by the bacterial flagellar motor. This motor is powered by the stator complex, MotA5 MotB2 , an heptameric complex which forms an ion channel which couples energy from the ion motive force to torque generation. Recent structural work revealed that stator complex consists of a ring of five MotA subunits which rotate around a central dimer of MotB subunits. Transmembrane (TM) domains TM3 and TM4 from MotA combine with the single TM domain from MotB to form two separate ion channels within this complex. Much is known about the ion binding site and ion specificity; however, to date, no modeling has been undertaken to explore the MotB-MotB dimer stability and the role of MotB conformational dynamics during rotation. Here, we modeled the central MotB dimer using coiled-coil engineering and modeling principles and calculated free energies to identify stable states in the operating cycle of the stator. We found three stable coiled-coil states with dimer interface angles of 28°, 56°, and 64°. We tested the effect of strategic mutagenesis on the comparative energy of the states and correlated motility with a specific hierarchy of stability between the three states. In general, our results indicate agreement with existing models describing a 36° rotation step of the MotA pentameric ring during the power stroke and provide an energetic basis for the coordinated rotation of the central MotB dimer based on coiled-coil modeling.
Asunto(s)
Proteínas Bacterianas , Flagelos , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Flagelos/química , Flagelos/metabolismo , Bacterias/metabolismo , Dominios Proteicos , Sitios de UniónRESUMEN
The design of protein interaction interfaces is a cornerstone of synthetic biology, where they can be used to promote the association of protein subunits into active molecular complexes or into protein nanostructures. In nature, protein interactions can be modulated by post-translational modifications (PTMs) that modify the protein interfaces with the addition and removal of various chemical groups. PTMs thus represent a means to gain control over protein interactions, yet they have seldom been considered in the design of synthetic proteins. Here, we explore the potential of a reversible PTM, serine phosphorylation, to modulate the interactions between peptides. We designed a series of interacting peptide pairs, including heterodimeric coiled coils, that contained one or more protein kinase A (PKA) recognition motifs. Our set of peptide pairs comprised interactions ranging from nanomolar to micromolar affinities. Mass spectrometry analyses showed that all peptides were excellent phosphorylation substrates of PKA, and subsequent phosphate removal could be catalyzed by lambda protein phosphatase. Binding kinetics measurements performed before and after treatment of the peptides with PKA revealed that phosphorylation of the target serines affected both the association and dissociation rates of the interacting peptides. We observed both the strengthening of interactions (up to an 11-fold decrease in Kd) and the weakening of interactions (up to a 180-fold increase in Kd). De novo-designed PTM-modulated interfaces will be useful to control the association of proteins in biological systems using protein-modifying enzymes, expanding the paradigm of self-assembly to encompass controlled assembly of engineerable protein complexes.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Dimerización , Cinética , Péptidos/análisis , Péptidos/química , Fosforilación , Unión Proteica , Serina/metabolismo , Resonancia por Plasmón de Superficie , Espectrometría de Masas en TándemRESUMEN
The transfer of electrons through protein complexes is central to cellular respiration. Exploiting proteins for charge transfer in a controllable fashion has the potential to revolutionize the integration of biological systems and electronic devices. Here we characterize the structure of an ultrastable protein filament and engineer the filament subunits to create electronically conductive nanowires under aqueous conditions. Cryoelectron microscopy was used to resolve the helical structure of gamma-prefoldin, a filamentous protein from a hyperthermophilic archaeon. Conjugation of tetra-heme c3-type cytochromes along the longitudinal axis of the filament created nanowires capable of long-range electron transfer. Electrochemical transport measurements indicated networks of the nanowires capable of conducting current between electrodes at the redox potential of the cytochromes. Functionalization of these highly engineerable nanowires with other molecules, such as redox enzymes, may be useful for bioelectronic applications.
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Metaloproteínas , Nanocables , Microscopía por Crioelectrón , Conductividad Eléctrica , Transporte de ElectrónRESUMEN
The field of proteomics is increasingly concerned with the diversity and functional relevance of protein modifications. Differential ion mobility spectrometry (DMS) is emerging as a tool to detect and quantify additional peptide and protein species that are difficult to analyse with conventional instrumental methods. In this review, recent advances in DMS are discussed, with a focus on the different types of DMS instruments now available to researchers in proteomics. Furthermore, the combination of DMS with mass spectrometry (MS) for increased proteome coverage and the targeted analysis of modification patterns on single proteins is highlighted. Frontier areas of research, such as the analysis of intact proteoforms, and future directions for the implementation of DMS in proteomics are also discussed.
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Espectrometría de Movilidad Iónica/métodos , Espectrometría de Masas/métodos , Proteínas/análisis , Proteoma/análisisRESUMEN
Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme-substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.