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1.
Plant Cell ; 35(12): 4347-4365, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37713604

RESUMO

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citoplasma/metabolismo , Parede Celular/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Raízes de Plantas , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo
2.
Nano Lett ; 23(13): 6259-6268, 2023 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-37141711

RESUMO

Amyloid-ß (Aß) aggregation intermediates, including oligomers and protofibrils (PFs), have attracted attention as neurotoxic aggregates in Alzheimer's disease. However, due to the complexity of the aggregation pathway, the structural dynamics of aggregation intermediates and how drugs act on them have not been clarified. Here we used high-speed atomic force microscopy to observe the structural dynamics of Aß42 PF at the single-molecule level and the effect of lecanemab, an anti-Aß PF antibody with the positive results from Phase 3 Clarity AD. PF was found to be a curved nodal structure with stable binding angle between individual nodes. PF was also a dynamic structure that associates with other PF molecules and undergoes intramolecular cleavage. Lecanemab remained stable in binding to PFs and to globular oligomers, inhibiting the formation of large aggregates. These results provide direct evidence for a mechanism by which antibody drugs interfere with the Aß aggregation process.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Microscopia de Força Atômica , Fragmentos de Peptídeos
3.
Methods ; 197: 4-12, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34107352

RESUMO

The structural dynamics of the amyloid protein aggregation process are associated with neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. High-speed atomic force microscopy (HS-AFM) is able to visualize the structural dynamics of individual aggregate species that otherwise cannot be distinguished. HS-AFM observations also detect impurities in the sample, and thus, experiments require relatively high sample purity. To derive valid information regarding the structural dynamics of the sample from the high-speed AFM images, a correction of the influence caused by the drift of the stage (scanner) from all frames is required. However, correcting the HS-AFM videos that consist of a large number of images requires significant effort. Here, using HS-AFM observation of α-synuclein fibril elongation as an example, we propose an HS-AFM image processing procedure to correct stage drift in the x-, y-, and z-directions with the free software ImageJ. ImageJ with default settings and our plugins attached to this article can process and analyze image stacks, which allow users to easily detect and show the temporal change in sample structures. This processing method can be automatically applied to numerous HS-AFM videos by batch processing with a series of ImageJ macrofunctions.


Assuntos
Doença de Alzheimer , Amiloide , Humanos , Microscopia de Força Atômica/métodos , Software
4.
Proc Natl Acad Sci U S A ; 117(14): 7831-7836, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213585

RESUMO

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.


Assuntos
Amiloide/ultraestrutura , Microscopia de Força Atômica , Fatores de Terminação de Peptídeos/ultraestrutura , Proteínas Priônicas/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Amiloide/genética , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Proteínas Priônicas/genética , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
5.
Int J Mol Sci ; 24(19)2023 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-37834298

RESUMO

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle.


Assuntos
Simulação de Dinâmica Molecular , Dobramento de Proteína , Animais , Raios X , Chaperoninas do Grupo II/química , Chaperoninas do Grupo II/metabolismo , Chaperoninas/metabolismo , Trifosfato de Adenosina/metabolismo , Nucleotídeos , Chaperonina com TCP-1/química , Conformação Proteica , Mamíferos/metabolismo
6.
Semin Cell Dev Biol ; 73: 132-144, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28739341

RESUMO

The amazing capacity of atomic force microscope to let us touch the molecular and cellular level samples with a sharp probe stimulated its application to bio-medical field among others. In addition to topographical imaging of the sample surface, a direct mechanical manipulation has attracted innovative minds to develop new methodologies aiming at direct handling of proteins, DNA/RNA, and cells. Measurement of their mechanical properties brought about a vivid picture of their physical nature. Direct handling of individual molecules and cells prompted development of nano-medical applications. This short review summarized recent application of AFM for measurement of mechanical properties of biological samples and attempts to perform direct manipulations of nano-medicine.


Assuntos
DNA/ultraestrutura , Microscopia de Força Atômica , Nanotecnologia , Proteínas/ultraestrutura , RNA/ultraestrutura , DNA/química , Proteínas/química , RNA/química
7.
FASEB J ; 33(8): 9220-9234, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31084283

RESUMO

Amyloid ß-protein (Aß) molecules tend to aggregate and subsequently form low MW (LMW) oligomers, high MW (HMW) aggregates such as protofibrils, and ultimately fibrils. These Aß species can generally form amyloid plaques implicated in the neurodegeneration of Alzheimer disease (AD), but therapies designed to reduce plaque load have not demonstrated clinical efficacy. Recent evidence implicates amyloid oligomers in AD neuropathology, but the precise mechanisms are uncertain. We examined the mechanisms of neuronal dysfunction from HMW-Aß1-42 exposure by measuring membrane integrity, reactive oxygen species (ROS) generation, membrane lipid peroxidation, membrane fluidity, intracellular calcium regulation, passive membrane electrophysiological properties, and long-term potentiation (LTP). HMW-Aß1-42 disturbed membrane integrity by inducing ROS generation and lipid peroxidation, resulting in decreased membrane fluidity, intracellular calcium dysregulation, depolarization, and impaired LTP. The damaging effects of HMW-Aß1-42 were significantly greater than those of LMW-Aß1-42. Therapeutic reduction of HMW-Aß1-42 may prevent AD progression by ameliorating direct neuronal membrane damage.-Yasumoto, T., Takamura, Y., Tsuji, M., Watanabe-Nakayama, T., Imamura, K., Inoue, H., Nakamura, S., Inoue, T., Kimura, A., Yano, S., Nishijo, H., Kiuchi, Y., Teplow, D. B., Ono, K. High molecular weight amyloid ß1-42 oligomers induce neurotoxicity via plasma membrane damage.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Cálcio/metabolismo , Linhagem Celular Tumoral , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fluidez de Membrana , Microscopia de Força Atômica , Peso Molecular , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismo
8.
Int J Mol Sci ; 21(12)2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32560229

RESUMO

Individual Alzheimer's disease (AD) patients have been shown to have structurally distinct amyloid-ß (Aß) aggregates, including fibrils, in their brain. These findings suggest the possibility of a relationship between AD progression and Aß fibril structures. Thus, the characterization of the structural dynamics of Aß could aid the development of novel therapeutic strategies and diagnosis. Protein structure and dynamics have typically been studied separately. Most of the commonly used biophysical approaches are limited in providing substantial details regarding the combination of both structure and dynamics. On the other hand, high-speed atomic force microscopy (HS-AFM), which simultaneously visualizes an individual protein structure and its dynamics in liquid in real time, can uniquely link the structure and the kinetic details, and it can also unveil novel insights. Although amyloidogenic proteins generate heterogeneously aggregated species, including transient unstable states during the aggregation process, HS-AFM elucidated the structural dynamics of individual aggregates in real time in liquid without purification and isolation. Here, we review and discuss the HS-AFM imaging of amyloid aggregation and strategies to optimize the experiments showing findings from Aß and amylin, which is associated with type II diabetes, shares some common biological features with Aß, and is reported to be involved in AD.


Assuntos
Peptídeos beta-Amiloides/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Imagem Molecular/métodos , Doença de Alzheimer/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Microscopia de Força Atômica , Agregados Proteicos , Conformação Proteica , Estabilidade Proteica
9.
Proc Natl Acad Sci U S A ; 113(21): 5835-40, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27162352

RESUMO

Aggregation of amyloidogenic proteins into insoluble amyloid fibrils is implicated in various neurodegenerative diseases. This process involves protein assembly into oligomeric intermediates and fibrils with highly polymorphic molecular structures. These structural differences may be responsible for different disease presentations. For this reason, elucidation of the structural features and assembly kinetics of amyloidogenic proteins has been an area of intense study. We report here the results of high-speed atomic force microscopy (HS-AFM) studies of fibril formation and elongation by the 42-residue form of the amyloid ß-protein (Aß1-42), a key pathogenetic agent of Alzheimer's disease. Our data demonstrate two different growth modes of Aß1-42, one producing straight fibrils and the other producing spiral fibrils. Each mode depends on initial fibril nucleus structure, but switching from one growth mode to another was occasionally observed, suggesting that fibril end structure fluctuated between the two growth modes. This switching phenomenon was affected by buffer salt composition. Our findings indicate that polymorphism in fibril structure can occur after fibril nucleation and is affected by relatively modest changes in environmental conditions.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/ultraestrutura , Fragmentos de Peptídeos/química , Amiloide/química , Peptídeos beta-Amiloides/síntese química , Soluções Tampão , Humanos , Microscopia de Força Atômica , Fragmentos de Peptídeos/síntese química , Cloreto de Potássio/química , Conformação Proteica , Cloreto de Sódio/química , Soluções , Propriedades de Superfície
12.
Nanomedicine ; 13(7): 2351-2357, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28756092

RESUMO

We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A2 was made to contact the cell membrane. A small dark hole (4-14 µm2 in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014µm2s-1. The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2).


Assuntos
Membrana Celular/patologia , Fibras de Estresse/patologia , Cicatrização , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células HeLa , Humanos , Microscopia de Força Atômica , Paxilina/análise , Paxilina/metabolismo , Análise de Célula Única , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura
13.
Proc Natl Acad Sci U S A ; 108(44): 17951-6, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21997211

RESUMO

F(1)-ATPase is a nanosized biological energy transducer working as part of F(o)F(1)-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F(1)-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F(1)-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of F(o)F(1)-ATP synthase. We found that the maximum work performed by F(1)-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F(1)-ATPase.


Assuntos
ATPases Translocadoras de Prótons/metabolismo , Termodinâmica , Trifosfato de Adenosina/metabolismo , Hidrólise
14.
Microscopy (Oxf) ; 71(3): 133-141, 2022 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-35253856

RESUMO

The assembly of misfolded proteins into amyloid fibrils is associated with amyloidosis, including neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases. The self-propagation of amyloid fibrils is widely observed in the aggregation pathways of numerous amyloidogenic proteins. This propensity with plasticity in primary nucleation allows amyloid fibril polymorphism, which is correlated with the pathology/phenotypes of patients. Because the interference with the nucleation and replication processes of amyloid fibrils can alter the amyloid structure and the outcome of the disease, these processes can be a target for developing clinical drugs. Single-molecule observations of amyloid fibril replication can be an experimental system to provide the kinetic parameters for simulation studies and confirm the effect of clinical drugs. Here, we review the single-molecule observation of the amyloid fibril replication process using fluorescence microscopy and time-lapse atomic force microscopy, including high-speed atomic force microscopy. We discussed the amyloid fibril replication process and combined single-molecule observation results with molecular dynamics simulations.


Assuntos
Amiloide , Simulação de Dinâmica Molecular , Amiloide/química , Humanos , Cinética , Microscopia de Força Atômica , Microscopia de Fluorescência
15.
J Mol Biol ; 434(2): 167385, 2022 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-34883118

RESUMO

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in ß-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin's C terminus (residues 31-37) that belongs to one of the two ß-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Maleatos/química , Conformação Molecular , Estireno/química , Amiloide/química , Animais , Simulação por Computador , Diabetes Mellitus Tipo 2 , Fluorescência , Humanos , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos , Espectroscopia de Infravermelho com Transformada de Fourier , Estirenos/química , Peixe-Zebra
16.
Biophys J ; 100(3): 564-572, 2011 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-21281570

RESUMO

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.


Assuntos
Fibroblastos/citologia , Microscopia de Força Atômica/métodos , Estresse Mecânico , Animais , Células Cultivadas , Citocalasina D/farmacologia , Elasticidade/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Ratos , Viscosidade/efeitos dos fármacos
17.
Neurochem Int ; 151: 105208, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34655726

RESUMO

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder and is characterized by major pathological hallmarks in the brain, including plaques composed of amyloid ß-protein (Aß) and neurofibrillary tangles of tau protein. Genetic studies, biochemical data, and animal models have suggested that Aß is a critical species in the pathogenesis of AD. Aß molecules aggregate to form oligomers, protofibrils (PFs), and mature fibrils. Because of their instability and structural heterogeneity, the misfolding and aggregation of Aß is a highly complex process, leading to a variety of aggregates with different structures and morphologies. However, the elucidation of Aß molecules is essential because they are believed to play an important role in AD pathogenesis. Recent combination studies using nuclear magnetic resonance (NMR) and cryo-electron microscopy (cryo-EM) have primarily revealed more detailed information about their aggregation process, including fibril extension and secondary nucleation, and the structural polymorphism of the fibrils under a variety of some conditions, including the actual brain. This review attempts to summarize the existing information on the major properties of the structure and aggregation of Aß.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Emaranhados Neurofibrilares/metabolismo , Placa Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Emaranhados Neurofibrilares/patologia , Placa Amiloide/patologia , Proteínas tau/metabolismo
18.
J Phys Chem Lett ; 12(15): 3837-3846, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33852305

RESUMO

DNA-histone interaction is always perturbed by epigenetic regulators to regulate gene expression. Direct visualization of this interaction is yet to be achieved. By using high-speed atomic force microscopy (HS-AFM), we have observed the dynamic DNA-histone H2A interaction. HS-AFM movies demonstrate the globular core and disordered tail of H2A. DNA-H2A formed the classic "beads-on-string" conformation on poly-l-lysine (PLL) and lipid substrates. Notably, a short-linearized double-stranded DNA (dsDNA), resembling an inchworm, wrapped around a single H2A protein only observed on the lipid substrate. Such a phenomenon does not occur for plasmid DNA or linearized long dsDNA on the same substrate. Strong adsorption of PLL substrate resulted in poor dynamic DNA-H2A interaction. Nonetheless, short-linearized dsDNA-H2A formed stable wrapping with a "diamond ring" topology on the PLL substrate. Reversible liquid-liquid phase separation (LLPS) of the DNA-H2A aggregate was visualized by manipulating salt concentrations. Collectively, our study suggest that HS-AFM is feasible for investigating epigenetically modified DNA-histone interactions.


Assuntos
DNA/química , Chaperonas de Histonas/química , Histonas/química , Microscopia de Força Atômica
19.
J Extracell Vesicles ; 10(14): e12170, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34874124

RESUMO

SARS-CoV-2 spike protein (S) binds to human angiotensin-converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high-speed atomic force microscopy (HS-AFM) for real-time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor-binding domain (RBD), and pH/temperature-induced conformational change of S. S in an S-ACE2 complex appears as an all-RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S-hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2-expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real-time visualization of various entry inhibitors, neutralizing antibodies, and sEV-based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS-CoV-2 spike and its interaction with receptor ACE2 and sEV-based decoy in real time using HS-AFM.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Temperatura , Internalização do Vírus
20.
Phys Rev Lett ; 104(19): 198103, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20867002

RESUMO

Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method. We show that the sum of the heat flow through the probe's rotational degree of freedom and the work against an external load is almost equal to the free energy change per a single ATP hydrolysis under various conditions. This implies that F1-ATPase works at an efficiency of nearly 100% in a thermally fluctuating environment.


Assuntos
Ensaios Enzimáticos/métodos , ATPases Translocadoras de Prótons/metabolismo , Bacillus/enzimologia , Eletricidade , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hidrólise , ATPases Translocadoras de Prótons/química , Rotação , Termodinâmica
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