Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 40
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biomacromolecules ; 22(10): 4146-4154, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34499838

RESUMO

Artificial protein cages have potential as programmable, protective carriers of fragile macromolecules to cells. While natural cages and VLPs have been extensively exploited, the use of artificial cages to deliver active proteins to cells has not yet been shown. TRAP-cage is an artificial protein cage with an unusual geometry and extremely high stability, which can be triggered to break apart in the presence of cellular reducing agents. Here, we demonstrate that TRAP-cage can be filled with a protein cargo and decorated with a cell-penetrating peptide, allowing it to enter cells. Tracking of both the TRAP-cage and the cargo shows that the protein of interest can be successfully delivered intracellularly in the active form. These results provide a valuable proof of concept for the further development of TRAP-cage as a delivery platform.


Assuntos
Nanotecnologia , Proteínas , Humanos , Conformação Proteica , Proteínas/química
2.
Nanoscale ; 13(27): 11932-11942, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34195748

RESUMO

Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium Thermotoga maritima (TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like Archeoglobus fulgidus ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg2+). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.


Assuntos
Ferritinas , Ferro , Ferritinas/genética , Ferro/metabolismo , Thermotoga maritima
3.
Nucleic Acids Res ; 49(3): 1581-1596, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33434265

RESUMO

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Inibidores da Topoisomerase II/toxicidade , Trifosfato de Adenosina/metabolismo , Bacteriocinas/toxicidade , Ciprofloxacina/toxicidade , DNA/metabolismo , Escherichia coli/enzimologia , Hidrólise , Compostos Orgânicos/toxicidade , Xanthomonas
4.
Methods Mol Biol ; 2208: 69-80, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32856256

RESUMO

DNA origami is a powerful technique, which allows virtually limitless 2D or 3D nanostructure designs to be constructed from DNA strands. Such nanostructures can even include programmable nanorobots, which are able to respond to the environment in predetermined ways. DNA aptamers hold particular promise as interfaces, which can enable proteins, peptides, and other non-nucleic acid biomolecules to trigger conformational changes in DNA nanostructures for diagnostic, biosensing, or therapeutic applications. Here, we provide the methodology for FRET-mediated observation of aptamer-triggered conformational change in a DNA origami box nanostructure. The method described can, in principle, be adapted to a wide variety of experimental circumstances where the DNA nanostructure conformational change is mediated by molecular or environmental cues.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência/métodos , Nanoestruturas/química , Proteínas/química , Aptâmeros de Nucleotídeos/química , Nanotecnologia/métodos , Conformação de Ácido Nucleico
5.
Methods Mol Biol ; 2208: 123-133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32856259

RESUMO

Protein and peptide cages are nanoscale containers, which are of particular interest in nanoscience due to their well-defined dimensions and enclosed central cavities that can be filled with material that is protected from the outside environment. Ferritin is a typical example of protein cage, formed by 24 polypeptide chains that self-assemble into a hollow, roughly spherical protein cage with external and internal diameters of approximately 12 nm and 8 nm, respectively. The interior cavity of ferritin provides a unique reaction vessel to carry out reactions separated from the exterior environment. In nature, the cavity is utilized for sequestration and biomineralization to render iron inert and safe by shielding from the external environment. Materials scientists have been inspired by this system and exploited a range of ferritin superfamily proteins as supramolecular templates to encapsulate cargoes ranging from cancer drugs to therapeutic proteins. Interesting possibilities arise if such containers can themselves be arranged into even higher-order structures such as crystalline arrays. Here, we describe how crystalline arrays of negatively charged ferritin protein cages can be built by taking advantage of electrostatic interactions with cationic gold nanoparticles.


Assuntos
Ferritinas/química , Análise Serial de Proteínas/métodos , Ouro/química , Ferro/química , Eletricidade Estática
6.
Trends Ecol Evol ; 35(5): 397-406, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32294421

RESUMO

Evolution requires self-replication. But, what was the very first self-replicator directly ancestral to all life? The currently favoured RNA World theory assigns this role to RNA alone but suffers from a number of seemingly intractable problems. Instead, we suggest that the self-replicator consisted of both peptides and nucleic acid strands. Such a nucleopeptide replicator is more feasible both in the light of the replication machinery currently found in cells and the complexity of the evolutionary path required to reach them. Recent theoretical and mathematical work supports this idea and provide a blueprint for future investigations.


Assuntos
Origem da Vida , RNA , Peptídeos/genética , RNA/genética
7.
Biochem J ; 477(7): 1345-1362, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32207815

RESUMO

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Simulação por Computador , DNA/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Fator Proteico para Inversão de Estimulação/genética , Expressão Gênica , Proteínas Mutantes/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica/genética , Proteínas Virais/química
8.
Nature ; 569(7756): 438-442, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31068697

RESUMO

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery1, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging2,3. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Aui-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.


Assuntos
Ouro/química , Proteínas/química , Microscopia Crioeletrônica , Cisteína/química , Mercúrio/química , Modelos Moleculares , Proteínas/ultraestrutura
9.
Nano Lett ; 19(6): 3918-3924, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31117758

RESUMO

Development of protein cages for encapsulation of active enzyme cargoes and their subsequent arrangement into a controllable three-dimensional array is highly desirable. However, cargo capture is typically challenging because of difficulties in achieving reversible assembly/disassembly of protein cages in mild conditions. Herein we show that by using an unusual ferritin cage protein that undergoes triggerable assembly under mild conditions, we can achieve reversible filling with protein cargoes including an active enzyme. We demonstrate that these filled cages can be arrayed in three-dimensional crystal lattices and have an additional chaperone-like effect, increasing both thermostability and enzymatic activity of the encapsulated enzyme.


Assuntos
Proteínas Arqueais/química , Archaeoglobus fulgidus/química , Proteínas de Bactérias/química , Preparações de Ação Retardada/química , Ferritinas/química , Thermotoga maritima/química , Sequência de Aminoácidos , Animais , Estabilidade Enzimática , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/química , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/química , Modelos Moleculares , Muramidase/administração & dosagem , Muramidase/química , Nanoestruturas/química , Ligação Proteica , Dobramento de Proteína
10.
Nanomedicine (Lond) ; 14(7): 911-925, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30901300

RESUMO

DNA nanotechnology research has long-held promise as a means of developing functional molecules capable of delivery to cells. Recent advances in DNA origami have begun to realize this potential but is still at the earliest stage and a number of hurdles remain. This review focuses on progress in addressing these hurdles and considers some of the challenges still outstanding. These include stability of such structures necessary to reach target cells after administration; methods of cell targeting and uptake; strategies to avoid or escape endosomes and techniques for achieving specific subcellular localization. Finally, the functionality that can be expected once DNA origami structures reach their final intracellular targets will be considered.


Assuntos
DNA/química , DNA/farmacologia , Nanopartículas/química , Animais , Sequência de Bases , Materiais Biocompatíveis/química , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , DNA/administração & dosagem , Terapia Genética/métodos , Humanos , Nanotecnologia/métodos , Conformação de Ácido Nucleico , Tamanho da Partícula , Propriedades de Superfície
11.
Chembiochem ; 19(18): 1900-1906, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-30007003

RESUMO

DNA aptamers are ideal tools to enable modular control of the dynamics of DNA nanostructures. For molecular recognition, they have a particular advantage over antibodies in that they can be integrated into DNA nanostructures in a bespoke manner by base pairing or nucleotide extension without any complex bioconjugation strategy. Such simplicity will be critical upon considering advanced therapeutic and diagnostic applications of DNA nanostructures. However, optimizing DNA aptamers for functional control of the dynamics of DNA nanostructure can be challenging. Herein, we present three considerations-shape, self-complementarity, and spatial flexibility-that should be paramount upon optimizing aptamer functionality. These lessons, learnt from the growing number of aptamer-nanostructure reports thus far, will be helpful for future studies in which aptamers are used to control the dynamics of nucleic acid nanostructures.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Nanoestruturas/química , Sequência de Bases , Modelos Moleculares , Nanotecnologia , Conformação de Ácido Nucleico
12.
Nanomedicine ; 14(4): 1161-1168, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29410111

RESUMO

DNA nanostructures can show dynamic responses to molecular triggers for a wide variety of applications. While DNA sequence signal triggers are now well-established, there is a critical need for a broader diversity of molecular triggers to drive dynamic responses in DNA nanostructures. DNA aptamers are ideal; they can both seamlessly integrate into DNA nanostructure scaffolds and transduce molecular recognition into functional responses. Here, we report construction and optimization of a DNA origami nanobox locked by a pair of DNA double strands where one strand is a DNA aptamer targeting the malaria biomarker protein Plasmodium falciparum lactate dehydrogenase. The protein acts as the key which enables box opening. We observe highly specific protein-mediated box opening by both transmission electron microscopy and fluorescence. Aptamer-enabled DNA boxes have significant potential for enabling direct responses to proteins and other biomolecules in nanoscale diagnostics, drug delivery and sensing devices.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Nanoestruturas/química , Animais , Biomarcadores/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Malária Falciparum/diagnóstico , Malária Falciparum/metabolismo , Microscopia Eletrônica de Transmissão , Nanoestruturas/ultraestrutura , Nanotecnologia , Proteínas de Protozoários/metabolismo
13.
Mol Biol Evol ; 35(2): 404-416, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29126321

RESUMO

Even the simplest organisms are too complex to have spontaneously arisen fully formed, yet precursors to first life must have emerged ab initio from their environment. A watershed event was the appearance of the first entity capable of evolution: the Initial Darwinian Ancestor. Here, we suggest that nucleopeptide reciprocal replicators could have carried out this important role and contend that this is the simplest way to explain extant replication systems in a mathematically consistent way. We propose short nucleic acid templates on which amino-acylated adapters assembled. Spatial localization drives peptide ligation from activated precursors to generate phosphodiester-bond-catalytic peptides. Comprising autocatalytic protein and nucleic acid sequences, this dynamical system links and unifies several previous hypotheses and provides a plausible model for the emergence of DNA and the operational code.


Assuntos
Modelos Químicos , Precursores de Ácido Nucleico/metabolismo , Nucleotídeos/metabolismo , Origem da Vida , Peptídeos/metabolismo , Polimerização
14.
Comput Struct Biotechnol J ; 15: 351-358, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28652896

RESUMO

Biological molecules, like organisms themselves, are subject to genetic drift and may even become "extinct". Molecules that are no longer extant in living systems are of high interest for several reasons including insight into how existing life forms evolved and the possibility that they may have new and useful properties no longer available in currently functioning molecules. Predicting the sequence/structure of such molecules and synthesizing them so that their properties can be tested is the basis of "molecular resurrection" and may lead not only to a deeper understanding of evolution, but also to the production of artificial proteins with novel properties and even to insight into how life itself began.

15.
Langmuir ; 32(23): 5899-908, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27181278

RESUMO

We present a simple synthesis of iron oxide nanotubes, grown under very mild conditions from a solution containing Fe(II) and Fe(III), on rod-shaped tobacco mosaic virus templates. Their well-defined shape and surface chemistry suggest that these robust bionanoparticles are a versatile platform for synthesis of small, thin mineral tubes, which was achieved efficiently. Various characterization tools were used to explore the iron oxide in detail: Electron microscopy (SEM, TEM), magnetometry (SQUID-VSM), diffraction (XRD, TEM-SAED), electron spectroscopies (EELS, EDX, XPS), and X-ray absorption (XANES with EXAFS analysis). They allowed determination of the structure, crystallinity, magnetic properties, and composition of the tubes. The protein surface of the viral templates was crucial to nucleate iron oxide, exhibiting analogies to biomineralization in natural compartments such as ferritin cages.


Assuntos
Compostos Férricos/química , Nanotubos/química , Vírus do Mosaico do Tabaco/química , Nanotubos/ultraestrutura , Vírus do Mosaico do Tabaco/ultraestrutura
16.
Sci Rep ; 6: 21266, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26891622

RESUMO

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology.


Assuntos
Aptâmeros de Nucleotídeos , Malária/diagnóstico , Malária/parasitologia , Proteínas de Protozoários/genética , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Biomarcadores , Humanos , Cinética , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Microscopia de Força Atômica , Modelos Moleculares , Plasmodium falciparum/genética , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
17.
PLoS One ; 10(11): e0142313, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26566222

RESUMO

Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.


Assuntos
DNA Girase/química , Malária Falciparum/parasitologia , Plasmodium falciparum/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , DNA Girase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Plasmodium falciparum/metabolismo , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência
18.
Nano Lett ; 15(2): 1331-5, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25559993

RESUMO

A cysteine-substituted mutant of the ring-shaped protein TRAP (trp-RNA binding attenuation protein) can be induced to self-assemble into large, monodisperse hollow spherical cages in the presence of 1.4 nm diameter gold nanoparticles. In this study we use high-speed atomic force microscopy (HS-AFM) to probe the dynamics of the structural changes related to TRAP interactions with the gold nanoparticle as well as the disassembly of the cage structure. The dynamic aggregation of TRAP protein in the presence of gold nanoparticles was observed, including oligomeric rearrangements, consistent with a role for gold in mediating intermolecular disulfide bond formation. We were also able to observe that the TRAP-cage is composed of multiple, closely packed TRAP rings in an apparently regular arrangement. A potential role for inter-ring disulfide bonds in forming the TRAP-cage was shown by the fact that ring-ring interactions were reversed upon the addition of reducing agent dithiothreitol. A dramatic disassembly of TRAP-cages was observed using HS-AFM after the addition of dithiothreitol. To the best of our knowledge, this is the first report to show direct high-resolution imaging of the disassembly process of a large protein complex in real time.


Assuntos
Microscopia de Força Atômica/métodos , Sondas Moleculares , Proteínas/química
19.
Appl Microbiol Biotechnol ; 98(23): 9545-60, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25343976

RESUMO

Pentapeptide repeats are a class of proteins characterized by the presence of multiple repeating sequences five amino acids in length. The sequences fold into a right-handed ß-helix with a roughly square-shaped cross section. Pentapeptide repeat proteins include a number of examples which are thought to function as structural mimics of DNA and act to competitively bind to the type II topoisomerase DNA gyrase, an important antibacterial target. DNA gyrase-targeting pentapeptide repeat proteins can both inhibit DNA gyrase-a potentially useful therapeutic property-and contribute to resistance to quinolone antibacterials (by acting to prevent them forming a lethal complex with the DNA and enzyme). Pentapeptide repeat proteins are therefore of wide interest not only because of their unusual structure, function, and potential as an antibacterial target, but also because knowledge of their mechanism of action may lead to both a greater understanding of the details of DNA gyrase function as well as being a useful template for the design of new DNA gyrase inhibitors. However, many puzzling aspects as to how these DNA mimics function and indeed even their ability to act as DNA mimics itself remains open to question. This review summarizes the current state of knowledge regarding pentapeptide repeat proteins, focusing on those that are thought to mimic DNA, and speculates on potential structure-function relationships which may account for their differing specificities.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Mimetismo Molecular , Sequências Repetitivas de Aminoácidos , Proteínas de Bactérias/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica
20.
PLoS One ; 9(8): e102454, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25083707

RESUMO

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redß recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.


Assuntos
Bacteriófagos/genética , Bacteriófagos/metabolismo , Família Multigênica , Recombinases/genética , Recombinases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Ordem dos Genes , Genoma Viral , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Recombinases/química , Alinhamento de Sequência , Proteínas Virais/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...