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1.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445134

RESUMO

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.


Assuntos
Antivirais/farmacologia , Infecções por Coxsackievirus/dietoterapia , Enterovirus Humano C/efeitos dos fármacos , Ácido N-Acetilneuramínico/farmacologia , Conjuntivite Hemorrágica Aguda/tratamento farmacológico , Conjuntivite Hemorrágica Aguda/metabolismo , Conjuntivite Hemorrágica Aguda/virologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Glucose/metabolismo , Humanos , Lectinas/metabolismo , Ligantes , Polissacarídeos/metabolismo , Receptores Virais/metabolismo
2.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
3.
Viruses ; 13(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064959

RESUMO

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.


Assuntos
Vetores Genéticos/genética , Vírus de Plantas/genética , Transdução Genética , Replicação Viral , Capsídeo/ultraestrutura , Vetores Genéticos/administração & dosagem , Vírus de Plantas/isolamento & purificação , Vírus de Plantas/ultraestrutura , Plantas/virologia , Tabaco/virologia
4.
Sci Rep ; 11(1): 9773, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963233

RESUMO

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.

5.
Commun Biol ; 4(1): 619, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031522

RESUMO

Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.


Assuntos
Proteínas do Capsídeo/metabolismo , Eucariotos/fisiologia , Folhas de Planta/virologia , Vírus de RNA/fisiologia , Tabaco/virologia , Vírion/fisiologia , Montagem de Vírus , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Quaternária de Proteína
6.
Sci Adv ; 7(3)2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33523887

RESUMO

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo-electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.

7.
Nat Commun ; 12(1): 44, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33398001

RESUMO

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the ß2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.


Assuntos
Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal , Polissacarídeos/metabolismo , Simbiose , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oligossacarídeos/química , Polissacarídeos/química , Conformação Proteica , Relação Estrutura-Atividade
8.
Nature ; 588(7838): 515-520, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33268888

RESUMO

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.


Assuntos
Microscopia Crioeletrônica , Miosina Tipo II/química , Miosina Tipo II/ultraestrutura , Animais , Ativação Enzimática , Estabilidade Enzimática , Modelos Moleculares , Músculo Liso/química , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Cadeias Leves de Miosina/ultraestrutura , Miosina Tipo II/metabolismo , Fosforilação , Domínios Proteicos , Perus
9.
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620

RESUMO

The ß-barrel assembly machinery (BAM) catalyses the folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The ß-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas , Lipídeos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Nanoestruturas , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteolipídeos/metabolismo
10.
PLoS Pathog ; 16(12): e1009146, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370422

RESUMO

Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses.


Assuntos
Proteínas do Capsídeo/metabolismo , Enterovirus/fisiologia , RNA Viral/genética , Montagem de Vírus/fisiologia , Sequência de Aminoácidos , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Enterovirus/ultraestrutura , RNA Viral/ultraestrutura
11.
Nat Struct Mol Biol ; 27(11): 1094, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33037421

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

12.
Nat Struct Mol Biol ; 27(11): 1048-1056, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32929282

RESUMO

Aggregation of the peptide hormone amylin into amyloid deposits is a pathological hallmark of type-2 diabetes (T2D). While no causal link between T2D and amyloid has been established, the S20G mutation in amylin is associated with early-onset T2D. Here we report cryo-EM structures of amyloid fibrils of wild-type human amylin and its S20G variant. The wild-type fibril structure, solved to 3.6-Å resolution, contains two protofilaments, each built from S-shaped subunits. S20G fibrils, by contrast, contain two major polymorphs. Their structures, solved at 3.9-Å and 4.0-Å resolution, respectively, share a common two-protofilament core that is distinct from the wild-type structure. Remarkably, one polymorph contains a third subunit with another, distinct, cross-ß conformation. The presence of two different backbone conformations within the same fibril may explain the increased aggregation propensity of S20G, and illustrates a potential structural basis for surface-templated fibril assembly.


Assuntos
Amiloide/genética , Diabetes Mellitus Tipo 2/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Amiloide/química , Amiloide/ultraestrutura , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Modelos Moleculares , Mutação Puntual , Conformação Proteica
13.
Structure ; 28(10): 1087-1100.e3, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32857965

RESUMO

Acinetobacter baumannii is a Gram-negative bacterium primarily associated with hospital-acquired, often multidrug-resistant (MDR) infections. The ribosome-targeting antibiotics amikacin and tigecycline are among the limited arsenal of drugs available for treatment of such infections. We present high-resolution structures of the 70S ribosome from A. baumannii in complex with these antibiotics, as determined by cryoelectron microscopy. Comparison with the ribosomes of other bacteria reveals several unique structural features at functionally important sites, including around the exit of the polypeptide tunnel and the periphery of the subunit interface. The structures also reveal the mode and site of interaction of these drugs with the ribosome. This work paves the way for the design of new inhibitors of translation to address infections caused by MDR A. baumannii.

14.
Nat Microbiol ; 5(8): 1016-1025, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32393857

RESUMO

Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a 'pedal bin' mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Periodontite/microbiologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/crescimento & desenvolvimento , Conformação Proteica
15.
PLoS Biol ; 18(3): e3000649, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32231352

RESUMO

Icosahedral viral capsids must undergo conformational rearrangements to coordinate essential processes during the viral life cycle. Capturing such conformational flexibility has been technically challenging yet could be key for developing rational therapeutic agents to combat infections. Noroviruses are nonenveloped, icosahedral viruses of global importance to human health. They are a common cause of acute gastroenteritis, yet no vaccines or specific antiviral agents are available. Here, we use genetics and cryo-electron microscopy (cryo-EM) to study the high-resolution solution structures of murine norovirus as a model for human viruses. By comparing our 3 structures (at 2.9- to 3.1-Å resolution), we show that whilst there is little change to the shell domain of the capsid, the radiating protruding domains are flexible, adopting distinct states both independently and synchronously. In doing so, the capsids sample a range of conformational space, with implications for maintaining virion stability and infectivity.


Assuntos
Capsídeo/química , Norovirus/química , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Dimerização , Temperatura Alta , Camundongos , Modelos Moleculares , Mutação , Norovirus/genética , Norovirus/patogenicidade , Domínios Proteicos , Células RAW 264.7 , Relação Estrutura-Atividade
16.
Curr Opin Struct Biol ; 60: 7-16, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31683043

RESUMO

In recent years our understanding of amyloid structure has been revolutionised by innovations in cryo-electron microscopy, electron diffraction and solid-state NMR. These techniques have yielded high-resolution structures of fibrils isolated from patients with neurodegenerative disease, as well as those formed from amyloidogenic proteins in vitro. The results not only show the expected cross-ß amyloid structure, but also reveal that the amyloid fold is unexpectedly diverse and complex. Here, we discuss this diversity, highlighting dynamic regions, ligand binding motifs, cavities, non-protein components, and structural polymorphism. Collectively, these variations combine to allow the generic amyloid fold to be realised in three dimensions in different ways, and this diversity may be related to the roles of fibrils in disease.


Assuntos
Amiloide/química , Amiloide/metabolismo , Animais , Doença , Humanos
17.
Nature ; 575(7783): 535-539, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723268

RESUMO

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.


Assuntos
Microscopia Crioeletrônica , Complexo Citocromos b6f/química , Complexo Citocromos b6f/ultraestrutura , Spinacia oleracea/química , Spinacia oleracea/ultraestrutura , Sítios de Ligação , Clorofila/química , Heme/química , Lipídeos/química , Modelos Moleculares , Oxirredução , Fotossíntese , Plastoquinona/química , Relação Estrutura-Atividade
18.
Structure ; 27(12): 1761-1770.e3, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31611039

RESUMO

The Luteoviridae are pathogenic plant viruses responsible for significant crop losses worldwide. They infect a wide range of food crops, including cereals, legumes, cucurbits, sugar beet, sugarcane, and potato and, as such, are a major threat to global food security. Viral replication is strictly limited to the plant vasculature, and this phloem limitation, coupled with the need for aphid transmission of virus particles, has made it difficult to generate virus in the quantities needed for high-resolution structural studies. Here, we exploit recent advances in heterologous expression in plants to produce sufficient quantities of virus-like particles for structural studies. We have determined their structures to high resolution by cryoelectron microscopy, providing the molecular-level insight required to rationally interrogate luteovirid capsid formation and aphid transmission, thereby providing a platform for the development of preventive agrochemicals for this important family of plant viruses.


Assuntos
Microscopia Crioeletrônica/métodos , Luteoviridae/ultraestrutura , Vírus de Plantas/ultraestrutura , Vírion/ultraestrutura , Sequência de Aminoácidos , Animais , Afídeos/fisiologia , Afídeos/virologia , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Regulação Viral da Expressão Gênica , Insetos Vetores/fisiologia , Insetos Vetores/virologia , Luteoviridae/genética , Luteoviridae/fisiologia , Floema/parasitologia , Floema/virologia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Conformação Proteica , Homologia de Sequência de Aminoácidos , Vírion/genética , Vírion/fisiologia
19.
Nucleic Acids Res ; 47(21): 11441-11451, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31642494

RESUMO

DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure-function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.


Assuntos
Precipitação Química , Colesterol/química , DNA de Cadeia Simples , Nanoestruturas/química , Nanotecnologia/métodos , Sequência de Bases/fisiologia , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico
20.
Front Plant Sci ; 10: 880, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354759

RESUMO

Virus-like particles (VLPs) of the fish virus, Atlantic Cod Nervous necrosis virus (ACNNV), were successfully produced by transient expression of the coat protein in Nicotiana benthamiana plants. VLPs could also be produced in transgenic tobacco BY-2 cells. The protein extracted from plants self-assembled into T = 3 particles, that appeared to be morphologically similar to previously analyzed NNV VLPs when analyzed by high resolution cryo-electron microscopy. Administration of the plant-produced VLPs to sea bass (Dicentrarchus labrax) showed that they could protect the fish against subsequent virus challenge, indicating that plant-produced vaccines may have a substantial future role in aquaculture.

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