Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 304
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 10(1): 2386, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160591

RESUMO

The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of graphene and embedded in vitreous ice. We are able to solve both the apo-SA and biotin-bound SA structures at near-atomic resolution using single-particle cryo-EM. We demonstrate that the method has the potential to determine the structures of molecules as small as 39 kDa.


Assuntos
Biotina/metabolismo , Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , Estreptavidina/ultraestrutura , Grafite , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Conformação Molecular , Estreptavidina/metabolismo
2.
Nat Commun ; 10(1): 2775, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235796

RESUMO

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.


Assuntos
Biocatálise , Endopeptidases/metabolismo , Proteínas de Plantas/metabolismo , Poliproteínas/síntese química , Engenharia de Proteínas/métodos , Estudos de Viabilidade , Microscopia de Força Atômica/métodos , Oldenlandia , Poliproteínas/genética , Poliproteínas/isolamento & purificação , Poliproteínas/ultraestrutura , Potyvirus , Desdobramento de Proteína , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Rubredoxinas/síntese química , Rubredoxinas/genética , Rubredoxinas/isolamento & purificação , Rubredoxinas/ultraestrutura , Imagem Individual de Molécula/métodos , Análise Espectral/métodos , Proteínas Virais
3.
Nat Commun ; 10(1): 2693, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217419

RESUMO

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.


Assuntos
Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Hipocampo/citologia , Humanos , Integrinas/metabolismo , Microscopia Intravital/métodos , Cinesina/genética , Cinesina/isolamento & purificação , Camundongos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Neurônios/citologia , Cultura Primária de Células , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
5.
Nat Commun ; 10(1): 2159, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089141

RESUMO

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Modelos Moleculares , DNA Helicases/isolamento & purificação , Proteínas de Drosophila/isolamento & purificação , Fenômenos Magnéticos , Pinças Ópticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
6.
Nat Commun ; 10(1): 2066, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061395

RESUMO

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.


Assuntos
Antígenos CD59/metabolismo , Membrana Celular/ultraestrutura , Complemento C9/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Multimerização Proteica , Membrana Celular/metabolismo , Complemento C5/metabolismo , Complemento C8/metabolismo , Humanos , Cinética , Microscopia de Força Atômica/métodos , Imagem Individual de Molécula/métodos
7.
Nat Commun ; 10(1): 2104, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068591

RESUMO

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.


Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
9.
Analyst ; 144(12): 3756-3764, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31070195

RESUMO

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and the determination of protein kinase activity plays a key role in the pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in a single living cell due to the shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in a single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring of the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitors and ultraviolet irradiation; we observed that endogenous RSK2 showed different behaviors in the cytoplasm and the nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.


Assuntos
Ensaios Enzimáticos/métodos , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Análise de Célula Única/métodos , Espectrometria de Fluorescência/métodos , Compostos de Boro/síntese química , Compostos de Boro/química , Carbocianinas/síntese química , Carbocianinas/química , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Imagem Individual de Molécula/métodos
10.
Nat Protoc ; 14(5): 1603-1633, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31019309

RESUMO

RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression.


Assuntos
Estabilidade de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/química , Imagem Individual de Molécula/métodos , Exorribonucleases , Microscopia de Fluorescência , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Nat Commun ; 10(1): 1855, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015431

RESUMO

DHX36 is a DEAH-box helicase that resolves parallel G-quadruplex structures formed in DNA and RNA. The recent co-crystal structure of DHX36 bound G4-DNA revealed an intimate contact, but did not address the role of ATP hydrolysis in G4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 displays ATP-independent unfolding of G4-RNA followed by ATP-dependent refolding, generating a highly asymmetric pattern of activity. Interestingly, DHX36 refolds G4-RNA in several steps, reflecting the discrete steps in forming the G4 structure. We show that the ATP-dependent activity of DHX36 arises from the RNA tail rather than the G4. Mutations that perturb G4 contact result in quick dissociation of the protein from RNA upon ATP hydrolysis, while mutations that interfere with binding the RNA tail induce dysregulated activity. We propose that the ATP-dependent activity of DHX36 may be useful for dynamically resolving various G4-RNA structures in cells.


Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Quadruplex G , Dobramento de RNA , RNA/metabolismo , RNA Helicases DEAD-box/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Microscopia de Fluorescência/métodos , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica/genética , RNA/química , Imagem Individual de Molécula/métodos
12.
Nat Methods ; 16(5): 387-395, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962624

RESUMO

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D and double-helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imagem Tridimensional/métodos , Imagem Individual de Molécula/métodos , Software , Algoritmos
13.
Nat Chem Biol ; 15(4): 401-409, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858596

RESUMO

We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.


Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Cor , Corantes Fluorescentes/síntese química , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Anticorpos de Domínio Único
14.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897814

RESUMO

Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Microscopia/métodos , Imagem Individual de Molécula/métodos , Simulação de Dinâmica Molecular , Multimerização Proteica
15.
Proc Natl Acad Sci U S A ; 116(13): 6091-6100, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850524

RESUMO

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Recombinação Homóloga , Nucleossomos/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos
16.
Nat Commun ; 10(1): 1232, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874551

RESUMO

Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.


Assuntos
Corantes Fluorescentes/química , Microscopia Intravital/métodos , Sondas Moleculares/química , Neurônios/química , Vesículas Sinápticas/química , Corantes Fluorescentes/efeitos da radiação , Células HeLa , Humanos , Isomerismo , Luz , Microscopia de Fluorescência/métodos , Sondas Moleculares/efeitos da radiação , Neurônios/citologia , Neurônios/metabolismo , Fotodegradação , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência , Vesículas Sinápticas/metabolismo , Imagem com Lapso de Tempo/métodos
17.
Nat Commun ; 10(1): 1172, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862823

RESUMO

The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09 ns (6% of unquenched brightness) and 0.21 ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.


Assuntos
Proteínas de Bactérias/metabolismo , Fotossíntese , Ficobilissomas/metabolismo , Synechocystis/fisiologia , Proteínas de Bactérias/química , Luz , Método de Monte Carlo , Ficobilissomas/isolamento & purificação , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência/métodos
18.
Nat Protoc ; 14(4): 1130-1168, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30903110

RESUMO

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS2) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores' efficiency when used for desalination or reverse electrodialysis applications. The atomic thickness of MoS2 nanopores approaches the inter-base distance of DNA, creating a potential candidate for DNA sequencing. In terms of geometry, MoS2 nanopores have a well-defined vertical profile due to their atomic thickness, which eliminates any unwanted effects associated with uneven pore profiles observed in other materials. This protocol details all the necessary procedures for the fabrication of solid-state devices. We discuss different methods for transfer of monolayer MoS2, different approaches for the creation of nanopores, their applicability in detecting DNA translocations and the analysis of translocation data through open-source programming packages. We present anticipated results through the application of our nanopores in DNA translocations and osmotic power generation. The procedure comprises four parts: fabrication of devices (2-3 d), transfer of MoS2 and cleaning procedure (24 h), the creation of nanopores within MoS2 (30 min) and performing DNA translocations (2-3 h). We anticipate that our protocol will enable large-scale manufacturing of single-molecule-analysis devices as well as next-generation DNA sequencing.


Assuntos
Dissulfetos/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microtecnologia/métodos , Molibdênio/química , Nanoporos/ultraestrutura , Nanotecnologia/métodos , DNA/análise , DNA/genética , Diálise/instrumentação , Diálise/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Microtecnologia/instrumentação , Nanotecnologia/instrumentação , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos
19.
Molecules ; 24(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704053

RESUMO

The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein⁻DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise individual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.


Assuntos
DNA/química , Imagem Individual de Molécula , Fenômenos Biofísicos , DNA/genética , DNA/ultraestrutura , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Microscopia de Fluorescência , Polímeros , Análise de Sequência de DNA , Imagem Individual de Molécula/métodos
20.
Mol Cell ; 73(5): 1015-1027.e7, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30711376

RESUMO

TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated somatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for T cell-based immunotherapy.


Assuntos
Imunidade Adaptativa , Antígeno HLA-A2/imunologia , Mecanotransdução Celular , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Células HEK293 , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Hibridomas , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem Individual de Molécula/métodos , Relação Estrutura-Atividade , Linfócitos T/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA