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1.
Nature ; 590(7846): 509-514, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33568813

RESUMEN

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.


Asunto(s)
Microscopía por Crioelectrón , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura , Escherichia coli/química , Canales Iónicos/metabolismo , Canales Iónicos/ultraestructura , Membranas Artificiales , Fosfatidilcolinas/metabolismo , Detergentes/farmacología , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Canales Iónicos/química , Canales Iónicos/genética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Modelos Moleculares , Mutación , Nanoestructuras/química , Nanoestructuras/ultraestructura , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacología , Conformación Proteica/efectos de los fármacos , beta-Ciclodextrinas/farmacología
2.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540888

RESUMEN

Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.


Asunto(s)
Pared Celular/química , Citocinas/metabolismo , Escherichia coli/química , Hierro/metabolismo , Lipopolisacáridos/farmacología , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Células THP-1/metabolismo , Ácidos Teicoicos/farmacología , Transporte Biológico , Receptor 1 de Quimiocinas CX3C/biosíntesis , Receptor 1 de Quimiocinas CX3C/genética , Quimiocina CX3CL1/metabolismo , Citocinas/biosíntesis , Citosol/metabolismo , Ferritinas/biosíntesis , Ferritinas/genética , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Hepcidinas/biosíntesis , Hepcidinas/genética , Humanos , Mitocondrias/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/genética , Células THP-1/efectos de los fármacos
3.
Nat Commun ; 12(1): 369, 2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33446644

RESUMEN

Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of the most widely disseminated pathways of polysaccharide biosynthesis is the inner membrane bound Wzy-dependent pathway. Here we present the 3.0 Å structure of the co-polymerase component of this pathway, WzzB from E. coli solved by single-particle cryo-electron microscopy. The overall architecture is octameric and resembles a box jellyfish containing a large bell-shaped periplasmic domain with the 2-helix transmembrane domain from each protomer, positioned 32 Å apart, encircling a large empty transmembrane chamber. This structure also reveals the architecture of the transmembrane domain, including the location of key residues for the Wzz-family of proteins and the Wzy-dependent pathway present in many Gram-negative bacteria, explaining several of the previous biochemical and mutational studies and lays the foundation for future investigations.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Polisacáridos Bacterianos/química , Microscopía por Crioelectrón , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismo , Modelos Moleculares , Polisacáridos Bacterianos/metabolismo , Regiones Promotoras Genéticas , Dominios Proteicos
4.
Mol Cell ; 81(4): 870-883.e10, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33453165

RESUMEN

The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Pliegue del ARN , ARN Bacteriano/química , Ribonucleasa P/química , Partícula de Reconocimiento de Señal/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , Ribonucleasa P/metabolismo , Partícula de Reconocimiento de Señal/metabolismo
5.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-33445776

RESUMEN

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide - endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.


Asunto(s)
Antígenos Bacterianos/química , Enterobacteriaceae/química , Lipopolisacáridos/química , Cromatografía/métodos , Fibras de la Dieta , Endotoxinas/química , Escherichia coli/química , Hidrólisis , Espectrometría de Masas/métodos , Polisacáridos/química , Shigella sonnei/química
6.
Methods Mol Biol ; 2199: 127-149, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33125648

RESUMEN

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.


Asunto(s)
Proteínas de Unión al ADN , Deuterio/química , Escherichia coli/química , Marcaje Isotópico , Proteínas Proto-Oncogénicas , Sistema Libre de Células/química , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Resonancia Magnética Nuclear Biomolecular , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
7.
Methods Mol Biol ; 2178: 81-91, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33128745

RESUMEN

Aqueous two-phase systems (ATPS) have been widely and successfully used in the purification of various biological macromolecules such as proteins, nucleic acids, antibiotics, and cell components. Interfacial precipitation of the product often results in lower recovery and selectivity of ATPS. Efficient resolubilization of the interfacial precipitate offers a way to improve the recovery as well as selectivity of ATPS systems.In this protocol, we describe a method for aqueous two-phase-assisted precipitation and resolubilization of the recombinant human Granulocyte Colony Stimulating Factor (GCSF) for its selective isolation from E. coli host cell proteins as well as nucleic acids. This platform purification can be applied to other cytokines as well as most of the hydrophobic proteins that partition into the hydrophobic PEG-rich top phase. Recoveries of up to 100% of the product along with reduction of levels of E. coli host cell proteins (from 250-500 to 10-15 ppm) and of nucleic acids (from 15-20 to 5-15 ng/mL) were observed.


Asunto(s)
Escherichia coli/química , Precipitación Fraccionada , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Factor Estimulante de Colonias de Granulocitos/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
8.
Methods Mol Biol ; 2178: 149-158, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33128749

RESUMEN

A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived Z-domain, and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchanger resin, and thereby allows solid-phase refolding of the target protein.Herein, protocols for purification of soluble Zbasic-tagged fusion proteins , as well as for integrated purification and solid-phase refolding of insoluble fusion proteins , are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.


Asunto(s)
Escherichia coli/química , Cuerpos de Inclusión/química , Proteínas Recombinantes de Fusión , Proteína Estafilocócica A , Cromatografía por Intercambio Iónico , Dominios Proteicos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteína Estafilocócica A/química , Proteína Estafilocócica A/aislamiento & purificación
9.
Methods Mol Biol ; 2178: 329-344, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33128759

RESUMEN

The bacterium Escherichia coli is still considered the first option as a microbial cell factory for recombinant protein production, and affinity chromatography is by far the preferred technique for initial purification after protein expression and cell lysis. In this chapter, we describe the methodology to express and purify recombinant proteins in E. coli tagged with the first two metal-binding proteins proposed as fusion partners. They are the small metal-binding protein SmbP and a mutant of the copper resistance protein CusF3H+. There are several advantages of using them as protein tags: they prevent the formation of inclusion bodies by increasing solubility of the target proteins, they enable purification by immobilized metal-affinity chromatography using Ni(II) ions with high purity, and because of their low molecular weights, excellent final yields are obtained for the target proteins after cleavage and removal of the protein tag. Here we also describe the protocol for the production of proteins in the periplasm of E. coli tagged with two SmbP variants that include the PelB or the TorA signal sequences for transport via the Sec or the Tat pathway, respectively. Based on these methods, we consider CusF3H+ and SmbP excellent alternatives as fusion proteins for the production of recombinant proteins in E. coli.


Asunto(s)
Cromatografía de Afinidad , Proteínas Transportadoras de Cobre , Proteínas de Escherichia coli , Escherichia coli/química , Níquel/química , Periplasma/química , Proteínas Transportadoras de Cobre/química , Proteínas Transportadoras de Cobre/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Periplasma/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación
10.
Methods Mol Biol ; 2178: 469-478, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33128766

RESUMEN

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.


Asunto(s)
Enzimas , Escherichia coli/química , Calor , Estabilidad de Enzimas , Enzimas/química , Enzimas/genética , Enzimas/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
11.
Biochim Biophys Acta Gen Subj ; 1865(1): 129762, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33053413

RESUMEN

BACKGROUND: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). METHODS: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. RESULTS: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. CONCLUSIONS: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. GENERAL SIGNIFICANCE: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Glucosa-6-Fosfato Isomerasa/metabolismo , Glutamato Descarboxilasa/metabolismo , Pirofosfatasa Inorgánica/metabolismo , Proteínas de la Membrana/metabolismo , Escherichia coli/química , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Fructosa-Bifosfato Aldolasa/química , Glucosa-6-Fosfato Isomerasa/química , Glutamato Descarboxilasa/química , Humanos , Pirofosfatasa Inorgánica/química , Cinética , Proteínas de la Membrana/química , Multimerización de Proteína
12.
Biochim Biophys Acta Gen Subj ; 1865(1): 129765, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33069832

RESUMEN

BACKGROUND: Heparin, a lifesaving blood thinner used in over 100 million surgical procedures worldwide annually, is currently isolated from over 700 million pigs and ~200 million cattle in slaughterhouses worldwide. Though animal-derived heparin has been in use over eight decades, it is a complex mixture that poses a risk for chemical adulteration, and its availability is highly vulnerable. Therefore, there is an urgent need in devising bioengineering approaches for the production of heparin polymers, especially low molecular weight heparin (LMWH), and thus, relying less on animal sources. One of the main challenges, however, is the rapid, cost-effective production of low molecular weight heparosan, a precursor of LMWH and size-defined heparosan oligosaccharides. Another challenge is N-sulfation of N-acetyl heparosan oligosaccharides efficiently, an essential modification required for subsequent enzymatic modifications, though chemical and enzymatic N-sulfation is effectively performed at the polymer level. METHODS: To devise a strategy to produce low molecular weight heparosan and heparosan oligosaccharides, several non-pathogenic E. coli strains are engineered by transforming the essential heparosan biosynthetic genes with or without the eliminase gene (elmA) from pathogenic E. coli K5. RESULTS: The metabolically engineered non-pathogenic strains are shown to produce ~5 kDa heparosan, a precursor for low molecular weight heparin, for the first time. Additionally, heparosan oligosaccharides of specific sizes ranging from tetrasaccharide to dodecasaccharide are directly generated, in a single step, from the recombinant bacterial strains that carry both heparosan biosynthetic genes and the eliminase gene. Various modifications, such as chemical N-sulfation of N-acetyl heparosan hexasaccharide following the selective protection of reducing end GlcNAc residue, enzymatic C5-epimerization of N-sulfo heparosan tetrasaccharide and complete 6-O sulfation of N-sulfo heparosan hexasaccharide, are shown to be feasible. CONCLUSIONS: We engineered non-pathogenic E. coli strains to produce low molecular weight heparosan and a range of size-specific heparosan oligosaccharides in a controlled manner through modulating culture conditions. We have also shown various chemical and enzymatic modifications of heparosan oligosaccharides. GENERAL SIGNIFICANCE: Heparosan is a precursor of heparin and the methods to produce low molecular weight heparosan is widely awaited. The methods described herein are promising and will pave the way for potential large scale production of low molecular weight heparin anticoagulants and bioactive heparin oligosaccharides in the coming decade.


Asunto(s)
Disacáridos/metabolismo , Escherichia coli/metabolismo , Ingeniería Metabólica , Oligosacáridos/metabolismo , Disacáridos/química , Disacáridos/genética , Escherichia coli/química , Escherichia coli/genética , Microbiología Industrial , Oligosacáridos/química , Oligosacáridos/genética
13.
Biosens Bioelectron ; 172: 112724, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33142197

RESUMEN

The uneven morphology and the trapped charges at the surface of the traditionally used supporting substrate-based 2D biosensors produces a scattering effect, which leads to a irregular signals from individually fabricated devices. Though suspended 2D channel material has the potential to overcome scattering effects from the substrates but achieving reliability and selectivity, have been limiting the using of this biosensor technology. Here, we have demonstrated nanogap electrodes fabrication by using the self-assembly technique, which provides suspension to the 2D-MoS2. These nano-spacing electrodes not only give suspension but also provide robustness strength to the atomic layer, which remains freestanding after coating of the Hafnium oxide (HfO2) as well as linkers and antibodies. For evaluating the electrical characteristics of suspended MoS2 FET, gating potential was applied through an electrolyte on the suspended MoS2 transistor. This helped in achieved a lower subthreshold swing 70 mV/dec and ON/OFF ratio 107. Later, pH detection was conducted at room temperature, which showed an impressive sensitivity of ~880 by changing 1 unit of pH. We have also successfully shown Escherichia coli (E. coli) bacteria sensing from the suspended MoS2 transistor by functionalizing dielectric layer with E. coli antibodies. The reported biosensor has shown the ~9% of conductance changes with a lower concentration of E. coli (10 CFU/mL; colony-forming unit per mL) as well as maintain the constant sensitivity in three fabricated devices. The obtained enhancement in the sensitivity of devices and its effect on biomolecules detection can be extened to other biomolecules and this type of architecture has the potential to detect COVID-19 viruses based biomolecules.


Asunto(s)
Técnicas Biosensibles/métodos , Disulfuros , Molibdeno , Nanoestructuras/química , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/estadística & datos numéricos , /virología , Materiales Biocompatibles Revestidos/química , Escherichia coli/química , Escherichia coli/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Microelectrodos , Microtecnología , Reproducibilidad de los Resultados , /aislamiento & purificación , Sensibilidad y Especificidad , Electricidad Estática , Volatilización
14.
Phys Chem Chem Phys ; 23(2): 1352-1362, 2021 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-33367433

RESUMEN

Adsorption processes are central to ionic transport in industrial and biological membrane systems. Multivalent cations modulate the conductive properties of nanofluidic devices through interactions with charged surfaces that depend principally on the ion charge number. Considering that ion channels are specialized valves that demand a sharp specificity in ion discrimination, we investigate the adsorption dynamics of trace amounts of different salts of trivalent cations in biological nanopores. We consider here OmpF from Escherichia coli, an archetypical protein nanopore, to probe the specificity of biological nanopores to multivalent cations. We systematically compare the effect of three trivalent electrolytes on OmpF current-voltage relationships and characterize the degree of rectification induced by each ion. We also analyze the open channel current noise to determine the existence of equilibrium/non-equilibrium mechanisms of ion adsorption and evaluate the extent of charge inversion through selectivity measurements. We show that the interaction of trivalent electrolytes with biological nanopores occurs via ion-specific adsorption yielding differential modulation of ion conduction and selectivity inversion. We also demonstrate the existence of non-equilibrium fluctuations likely related to ion-dependent trapping-detrapping processes. Our study provides fundamental information relevant to different biological and electrochemical systems where transport phenomena involve ion adsorption in charged surfaces under nanoscale confinement.


Asunto(s)
Complejos de Coordinación/química , Lantano/química , Nanoporos , Porinas/química , Espermidina/química , Adsorción , Cationes/química , Cobalto/química , Escherichia coli/química
15.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-33348897

RESUMEN

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N-hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom-up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.


Asunto(s)
Escherichia coli/química , Ésteres/química , Indicadores y Reactivos/química , Fragmentos de Péptidos/química , Proteoma/análisis , Succinimidas/química , Escherichia coli/metabolismo , Proteoma/química , Espectrometría de Masa por Ionización de Electrospray
16.
Molecules ; 25(24)2020 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-33371472

RESUMEN

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis.


Asunto(s)
Proteínas/análisis , Proteínas/química , Animales , Cinamatos/química , Corylus/química , Ácidos Cumáricos/química , Escherichia coli/química , Leche/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos
17.
Nature ; 584(7821): 479-483, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32788728

RESUMEN

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Asunto(s)
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidad , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencias de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Estabilidad de Enzimas , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Genes Esenciales , Hidrolasas/química , Hidrolasas/metabolismo , Lípido A/química , Lípido A/metabolismo , Lipopolisacáridos/biosíntesis , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Periplasma/química , Periplasma/metabolismo , Unión Proteica , Virulencia
18.
Proc Natl Acad Sci U S A ; 117(33): 19879-19887, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32747536

RESUMEN

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.


Asunto(s)
Escherichia coli/genética , ARN Bacteriano/química , ARN Ribosómico 23S/química , Ribosomas/genética , Escherichia coli/química , Escherichia coli/metabolismo , Evolución Molecular , Conformación de Ácido Nucleico , Filogenia , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , Ribosomas/química , Ribosomas/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(31): 18540-18549, 2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32675239

RESUMEN

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagen Individual de Molécula , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(31): 18737-18743, 2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32675245

RESUMEN

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral ß-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the ß-barrel assembly machine (BAM), which is composed of one OMP-BamA-and four lipoproteins-BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamA E470K , that bypasses the requirement for BamD. Although bamD::kan bamA E470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Membrana Externa Bacteriana , Proteínas de Escherichia coli , Mutación con Ganancia de Función/genética , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
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