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2.
PLoS Biol ; 16(6): e2006191, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29924793

RESUMO

Herpesviruses include many important human pathogens such as herpes simplex virus, cytomegalovirus, varicella-zoster virus, and the oncogenic Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus. Herpes virions contain a large icosahedral capsid that has a portal at a unique 5-fold vertex, similar to that seen in the tailed bacteriophages. The portal is a molecular motor through which the viral genome enters the capsid during virion morphogenesis. The genome also exits the capsid through the portal-vertex when it is injected through the nuclear pore into the nucleus of a new host cell to initiate infection. Structural investigations of the herpesvirus portal-vertex have proven challenging, owing to the small size of the tail-like portal-vertex-associated tegument (PVAT) and the presence of the tegument layer that lays between the nucleocapsid and the viral envelope, obscuring the view of the portal-vertex. Here, we show the structure of the herpes simplex virus portal-vertex at subnanometer resolution, solved by electron cryomicroscopy (cryoEM) and single-particle 3D reconstruction. This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures that occupy the sites normally taken by the penton and the Ta triplex. Our data revealed that the PVAT is composed of 10 copies of the C-terminal domain of pUL25, which are uniquely arranged as two tiers of star-shaped density. Our 3D reconstruction of the portal-vertex also shows that one end of the viral genome extends outside the portal in the manner described for some bacteriophages but not previously seen in any eukaryote viruses. Finally, we show that the viral genome is consistently packed in a highly ordered left-handed spool to form concentric shells of DNA. Our data provide new insights into the structure of a molecular machine critical to the biology of an important class of human pathogens.


Assuntos
Capsídeo/ultraestrutura , Herpesvirus Humano 1/ultraestrutura , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , DNA Viral/química , Genoma Viral , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Imageamento Tridimensional , Modelos Biológicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/ultraestrutura , Montagem de Vírus
3.
Science ; 360(6389)2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29650704

RESUMO

Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.


Assuntos
Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , Proteínas Motores Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Trifosfato de Adenosina/biossíntese , Microscopia Crioeletrônica , Lipídeos de Membrana/química , Membranas Mitocondriais/química , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Proteínas Motores Moleculares/ultraestrutura , Oligomicinas/química , Conformação Proteica , Subunidades Proteicas , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Imagem Individual de Molécula
4.
Biochem Biophys Res Commun ; 496(1): 12-17, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29294326

RESUMO

The bacterial flagellar motor rotates in both counterclockwise (CCW) and clockwise (CW) directions. FliG, FliM and FliN form the C ring on the cytoplasmic face of the MS ring made of a transmembrane protein, FliF. The C ring acts not only as a rotor but also as a switch of the direction of motor rotation. FliG consists of three domains: FliGN, FliGM and FliGC. FliGN directly binds to FliF. Intermolecular interactions between FliGM and FliGC drive FliG ring formation. FliGM is responsible for the interaction with FliM. FliGC is involved in the interaction with the stator protein MotA. Adaptive remodeling of the C ring occurs when the motor switches between the CCW and CW states. However, it remained unknown how. Here, we report the effects of a CW-locked deletion mutation (ΔPEV) in FliG of Thermotaoga maritia (Tm-FliG) on FliG-FliG and FliG-FliM interactions. The PEV deletion stabilized the intramolecular interaction between FliGM and FliGC, thereby suppressing the oligomerization of Tm-FliGMC in solution. This deletion also induced a conformational change of HelixMC connecting FliGM and FliGC to reduce the binding affinity of Tm-FliGMC for FliM. We will discuss adaptive remodeling of the C ring responsible for flagellar motor switching.


Assuntos
Proteínas de Bactérias/química , Flagelos/química , Proteínas Motores Moleculares/química , Movimento (Física) , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Proteínas Motores Moleculares/ultraestrutura , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
5.
Biochem Biophys Res Commun ; 495(3): 2145-2151, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29248727

RESUMO

There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.


Assuntos
Actinas/química , Proteínas de Arabidopsis/química , Proteínas Motores Moleculares/química , Movimento (Física) , Miosinas/química , Actinas/ultraestrutura , Proteínas de Arabidopsis/ultraestrutura , Ativação Enzimática , Proteínas Motores Moleculares/ultraestrutura , Miosinas/ultraestrutura , Ligação Proteica
6.
Biochem Biophys Res Commun ; 493(1): 388-392, 2017 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-28887032

RESUMO

Recent studies suggested a link between diversity of beta tubulin isotypes in microtubule structures and the regulatory roles that they play not only on microtubules' intrinsic dynamic, but also on the translocation characteristics of some of the molecular motors along microtubules. Remarkably, unlike porcine brain microtubules, MCF7 microtubules are structured from a different beta tubulin distribution. These types of cancer microtubules show a relatively stable and slow dynamic. In addition, the translocation parameters of some molecular motors are distinctly different along MCF7 as compared to those parameters on brain microtubules. It is known that the diversity of beta tubulin isotypes differ predominantly in the specifications and the electric charge of their carboxy-terminal tails. A key question is to identify whether the negative electrostatic charge of tubulin isotypes and, consequently, microtubules, can potentially be considered as one of the sources of functional differences in MCF7 vs. brain microtubules. We tested this possibility experimentally by monitoring the electro-orientation of these two types of microtubules inside a uniform electric field. Through this evaluation, we quantified and compared the average normalized polarization coefficient of MCF7 vs. Porcine brain microtubules. The higher value obtained for the polarization of MCF7 microtubules, which is associated to the higher negative charge of these types of microtubules, is significant as it can further explain the slow intrinsic dynamic that has been recently reported for single MCF7 microtubules in vitro. Furthermore, it can be potentially considered as a factor that can directly impact the translocation parameters of some molecular motors along MCF7 microtubules, by altering the mutual electrostatic interactions between microtubules and molecular motors.


Assuntos
Química Encefálica , Campos Eletromagnéticos , Microtúbulos/efeitos da radiação , Microtúbulos/ultraestrutura , Proteínas Motores Moleculares/efeitos da radiação , Proteínas Motores Moleculares/ultraestrutura , Eletricidade Estática , Animais , Humanos , Células MCF-7 , Microtúbulos/química , Proteínas Motores Moleculares/química , Movimento (Física) , Dosímetros de Radiação , Suínos
7.
Nat Commun ; 8: 15091, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28474682

RESUMO

Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward. The motor ATPase PilB powers pilus assembly. Here we report the structures of the core ATPase domains of Geobacter metallireducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMP-PNP, at 3.4 and 2.3 Å resolution, respectively. These structures reveal important differences in nucleotide binding between chains. Analysis of these differences reveals the sequential turnover of nucleotide, and the corresponding domain movements. Our data suggest a clockwise rotation of the central sub-pores of PilB, which through interactions with PilC, would support the assembly of a right-handed helical pilus. Our analysis also suggests a counterclockwise rotation of the C2 symmetric PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction.


Assuntos
Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/ultraestrutura , Adenilil Imidodifosfato/metabolismo , Proteínas de Bactérias/ultraestrutura , Proteínas de Fímbrias/ultraestrutura , Fímbrias Bacterianas/metabolismo , Proteínas Motores Moleculares/ultraestrutura , Oxirredutases/ultraestrutura , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Geobacter , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Nucleotídeos/metabolismo , Oxirredutases/metabolismo , Virulência
8.
Nat Commun ; 8: 14276, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-28120828

RESUMO

The bacterial flagellum is a motile organelle driven by a rotary motor, and its axial portions function as a drive shaft (rod), a universal joint (hook) and a helical propeller (filament). The rod and hook are directly connected to each other, with their subunit proteins FlgG and FlgE having 39% sequence identity, but show distinct mechanical properties; the rod is straight and rigid as a drive shaft whereas the hook is flexible in bending as a universal joint. Here we report the structure of the rod and comparison with that of the hook. While these two structures have the same helical symmetry and repeat distance and nearly identical folds of corresponding domains, the domain orientations differ by ∼7°, resulting in tight and loose axial subunit packing in the rod and hook, respectively, conferring the rigidity on the rod and flexibility on the hook. This provides a good example of versatile use of a protein structure in biological organisms.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Flagelos/fisiologia , Proteínas Motores Moleculares/química , Salmonella typhimurium/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Flagelos/ultraestrutura , Imageamento Tridimensional , Simulação de Acoplamento Molecular , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/ultraestrutura , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína/fisiologia , Salmonella typhimurium/citologia , Alinhamento de Sequência
9.
Proc Natl Acad Sci U S A ; 113(17): 4783-7, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27071081

RESUMO

Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Técnicas de Sonda Molecular , Fotodegradação , Rotação
10.
Nat Commun ; 7: 10323, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26744226

RESUMO

The actin cytoskeleton--a complex, nonequilibrium network consisting of filaments, actin-crosslinking proteins (ACPs) and motors--confers cell structure and functionality, from migration to morphogenesis. While the core components are recognized, much less is understood about the behaviour of the integrated, disordered and internally active system with interdependent mechano-chemical component properties. Here we use a Brownian dynamics model that incorporates key and realistic features--specifically actin turnover, ACP (un)binding and motor walking--to reveal the nature and underlying regulatory mechanisms of overarching cytoskeletal states. We generate multi-dimensional maps that show the ratio in activity of these microscopic elements determines diverse global stress profiles and the induction of nonequilibrium morphological phase transition from homogeneous to aggregated networks. In particular, actin turnover dynamics plays a prominent role in tuning stress levels and stabilizing homogeneous morphologies in crosslinked, motor-driven networks. The consequence is versatile functionality, from dynamic steady-state prestress to large, pulsed constrictions.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Motores Moleculares/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Citocalasina D/farmacologia , Humanos , Microscopia Confocal , Simulação de Dinâmica Molecular , Proteínas Motores Moleculares/efeitos dos fármacos , Proteínas Motores Moleculares/ultraestrutura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transição de Fase , Estresse Mecânico , Tiazolidinas/farmacologia , Imagem com Lapso de Tempo
11.
Nat Nanotechnol ; 11(2): 184-90, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26619152

RESUMO

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next-generation sensors, drug-delivery platforms and biological computing. Despite their exquisite programmability, DNA-based walkers are challenging to work with because of their low fidelity and slow rates (∼1 nm min(-1)). Here we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three orders of magnitude greater than the maximum for conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridize to a surface modified with complementary RNA; the motion is achieved through the addition of RNase H, which selectively hydrolyses the hybridized RNA. The spherical motors can move in a self-avoiding manner, and anisotropic particles, such as dimerized or rod-shaped particles, can travel linearly without a track or external force. We also show that the motors can be used to detect single nucleotide polymorphism by measuring particle displacement using a smartphone camera.


Assuntos
DNA/química , Proteínas Motores Moleculares/química , Nanotecnologia/métodos , Ribonuclease H/metabolismo , Computadores Moleculares , DNA/metabolismo , DNA/ultraestrutura , Sistemas de Liberação de Medicamentos , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/ultraestrutura , Ribonuclease H/química
12.
Nat Biotechnol ; 33(10): 1073-5, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26414351

RESUMO

Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.


Assuntos
DNA Helicases/química , DNA/química , Micromanipulação/métodos , Proteínas Motores Moleculares/química , Nanoporos/ultraestrutura , DNA/ultraestrutura , DNA Helicases/ultraestrutura , Módulo de Elasticidade , Teste de Materiais/métodos , Proteínas Motores Moleculares/ultraestrutura , Movimento (Física) , Nanotecnologia/métodos , Ligação Proteica , Estresse Mecânico
13.
Biosystems ; 132-133: 1-5, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25864376

RESUMO

Myosins are typical molecular motor proteins that convert the chemical energy from the ATP hydrolysis into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results already obtained, Masuda has proposed a hypothesis called the "Driven by Detachment" theory for the working principle of the myosins. This theory insists that the energy used during the power stroke of the myosins does not directly originate from the chemical energy of ATP, but is converted from the elastic energy within the molecule at the joint between the head and neck domains. One method for demonstrating the validity of this theory is a computational simulation using the molecular dynamics (MD) method. The MD software used was GROMACS. The target of the MD simulations was myosin subfragment-1 (S1), for which the initial structure was obtained from the Protein Data Bank entry 1M8Q. The AFM pull code of GROMACS was used to apply an external force of 17 pN at the end of the neck domain in the direction opposite to the power stroke to observe whether the myosin S1 takes the pre-power stroke conformation. The residues assumed to be engaged in the docking with an actin filament were fixed to the space. Starting from exactly the same initial position, 10 simulations were repeated by varying the random seeds for generating the initial velocities of the atoms. After 64ns of calculations, the myosin S1 took the conformation of the pre-power stroke state in which the neck domain was bent around the joint between the head and the neck domains. This result agrees with the prediction expected by the DbD theory, the validity of which may be established by conducting similar simulations for the other steps of the myosin working processes.


Assuntos
Actinas/química , Simulação de Dinâmica Molecular , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Subfragmentos de Miosina/química , Subfragmentos de Miosina/ultraestrutura , Actinas/ultraestrutura , Sítios de Ligação , Módulo de Elasticidade , Modelos Químicos , Movimento (Física) , Ligação Proteica , Conformação Proteica
14.
Nano Lett ; 15(4): 2456-61, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25736894

RESUMO

Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors.


Assuntos
DNA/química , Calefação/métodos , Imagem Molecular/métodos , Miosinas/química , Pinças Ópticas , Termografia/métodos , DNA/ultraestrutura , Ouro/química , Teste de Materiais/métodos , Nanopartículas Metálicas/química , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Técnicas de Sonda Molecular , Miosinas/isolamento & purificação , Temperatura
15.
Biomech Model Mechanobiol ; 14(5): 1107-17, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25676575

RESUMO

Kinesin is a motor protein that delivers cargo inside a cell. Kinesin has many different families, but they perform basically same function and have same motions. The walking motion of kinesin enables the cargo delivery inside the cell. Autoinhibition of kinesin is important because it explains how function of kinesin inside a cell is stopped. Former researches showed that tail binding is related to autoinhibition of kinesin. In this work, we performed normal mode analysis with elastic network model using different conformation of kinesin to determine the effect of tail binding by considering four models such as functional form, autoinhibited form, autoinhibited form without tail, and autoinhibited form with carbon structure. Our calculation of the thermal fluctuation and cross-correlation shows the change of tail-binding region in structural motion. Also strain energy of kinesin showed that elimination of tail binding effect leads the structure to have energetically similar behavior with the functional form.


Assuntos
Difosfato de Adenosina/química , Cinesina/química , Cinesina/ultraestrutura , Modelos Químicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Sítios de Ligação , Simulação por Computador , Módulo de Elasticidade , Transferência de Energia , Cinética , Proteínas Motores Moleculares/ultraestrutura , Movimento (Física) , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Temperatura
16.
Nanoscale ; 7(1): 82-5, 2015 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-25254951

RESUMO

A smart self-powered cargo delivery system that is composed of creatine phosphate kinase (CPK) microspheres, kinesins and microtubules is demonstrated. The CPK microsphere not only acts as an ATP generation and buffering system, but also as a carrier for cargo transport, thus realizing the easy loading and self-powered delivery of cargos at the same time.


Assuntos
Preparações de Ação Retardada/química , Cinesina/química , Microtúbulos/química , Proteínas Motores Moleculares/química , Nanocápsulas/química , Robótica/métodos , Cinesina/ultraestrutura , Teste de Materiais , Microtúbulos/ultraestrutura , Proteínas Motores Moleculares/ultraestrutura , Movimento (Física) , Nanocápsulas/ultraestrutura , Tamanho da Partícula
17.
Sci Rep ; 4: 7255, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25434968

RESUMO

Within living cells, the transport of cargo is accomplished by groups of molecular motors. Such collective transport could utilize mechanisms which emerge from inter-motor interactions in ways that are yet to be fully understood. Here we combined experimental measurements of two-kinesin transport with a theoretical framework to investigate the functional ramifications of inter-motor interactions on individual motor function and collective cargo transport. In contrast to kinesin's low sidestepping frequency when present as a single motor, with exactly two kinesins per cargo, we observed substantial motion perpendicular to the microtubule. Our model captures a surface-associated mode of kinesin, which is only accessible via inter-motor interference in groups, in which kinesin diffuses along the microtubule surface and rapidly "hops" between protofilaments without dissociating from the microtubule. Critically, each kinesin transitions dynamically between the active stepping mode and this weak surface-associated mode enhancing local exploration of the microtubule surface, possibly enabling cellular cargos to overcome macromolecular crowding and to navigate obstacles along microtubule tracks without sacrificing overall travel distance.


Assuntos
Cinesina/química , Microtúbulos/química , Modelos Químicos , Proteínas Motores Moleculares/química , Movimento (Física) , Simulação por Computador , Transferência de Energia , Cinesina/ultraestrutura , Microtúbulos/ultraestrutura , Modelos Moleculares , Proteínas Motores Moleculares/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Conformação Proteica
18.
Nanoscale ; 6(24): 15008-19, 2014 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-25367216

RESUMO

Synthetic molecular motors typically take nanometer-scale steps through rectification of thermal motion. Here we propose Inchworm, a DNA-based motor that employs a pronounced power stroke to take micrometer-scale steps on a time scale of seconds, and we design, fabricate, and analyze the nanofluidic device needed to operate the motor. Inchworm is a kbp-long, double-stranded DNA confined inside a nanochannel in a stretched configuration. Motor stepping is achieved through externally controlled changes in salt concentration (changing the DNA's extension), coordinated with ligand-gated binding of the DNA's ends to the functionalized nanochannel surface. Brownian dynamics simulations predict that Inchworm's stall force is determined by its entropic spring constant and is ∼ 0.1 pN. Operation of the motor requires periodic cycling of four different buffers surrounding the DNA inside a nanochannel, while keeping constant the hydrodynamic load force on the DNA. We present a two-layer fluidic device incorporating 100 nm-radius nanochannels that are connected through a few-nm-wide slit to a microfluidic system used for in situ buffer exchanges, either diffusionally (zero flow) or with controlled hydrodynamic flow. Combining experiment with finite-element modeling, we demonstrate the device's key performance features and experimentally establish achievable Inchworm stepping times of the order of seconds or faster.


Assuntos
DNA/química , DNA/ultraestrutura , Microfluídica/instrumentação , Nanotecnologia/instrumentação , Transdutores , Animais , Anelídeos/fisiologia , Biomimética/instrumentação , Difusão , Transferência de Energia , Desenho de Equipamento , Análise de Falha de Equipamento , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Movimento (Física) , Conformação de Ácido Nucleico , Estresse Mecânico
19.
Artigo em Inglês | MEDLINE | ID: mdl-25353516

RESUMO

Intracellular transport based on molecular motors and its regulation are crucial to the functioning of cells. Filamentary tracks of the cells are abundantly decorated with nonmotile microtubule-associated proteins, such as tau. Motivated by experiments on kinesin-tau interactions [Dixit et al., Science 319, 1086 (2008)] we developed a stochastic model of interacting single-headed motor proteins KIF1A that also takes into account the interactions between motor proteins and tau molecules. Our model reproduces experimental observations and predicts significant effects of tau on bound time and run length which suggest an important role of tau in regulation of kinesin-based transport.


Assuntos
Cinesina/química , Modelos Químicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas tau/química , Sítios de Ligação , Simulação por Computador , Cinesina/ultraestrutura , Cinética , Proteínas Motores Moleculares/ultraestrutura , Movimento (Física) , Ligação Proteica , Conformação Proteica , Proteínas tau/ultraestrutura
20.
Prog Biophys Mol Biol ; 115(2-3): 328-39, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25042577

RESUMO

A major motivation for the use of super-resolution imaging methods in the investigation of cardiac biophysics has been the insight from biophysical considerations and detailed mathematical modeling that the spatial structure and protein organisation at the scale of nanometres can have enormous implications for calcium signalling in cardiac muscle. We illustrate the use of dSTORM based super-resolution in optically thick (∼10 µm) tissue slices of rat ventricular tissue to visualize proteins at the cardiac Z-disk and compare those images with confocal (diffraction-limited) as well as electron microscopy (EM) data which still provides a benchmark in terms of resolution. α-actinin is an abundant protein target that effectively defines the Z-disk in striated muscle and provides a reference structure for other proteins at the Z-line and the transverse tubules. Using super-resolution imaging α-actinin labelling provides very detailed outlines of the contractile machinery which we have used to study the properties of Z-disks and the distribution of α-actinin itself. We determined the local diameters of the myo-fibrillar and non-myofibrillar space using α-actinin labelling. Comparison between confocal and super-resolution based myofibrillar masks suggested that super-resolution data was able to segment myofibrils accurately while confocal approaches were not always able to distinguish neighbouring myofibrillar bundles which resulted in overestimated diameters. The increased resolution of super-resolution methods provides qualitatively new information to improve our understanding of cardiac biophysics. Nevertheless, conventional diffraction-limited imaging still has an important role to play which we illustrate with correlative confocal and super-resolution data.


Assuntos
Actinina/ultraestrutura , Ventrículos do Coração/ultraestrutura , Aumento da Imagem/métodos , Microscopia de Fluorescência/métodos , Proteínas Motores Moleculares/ultraestrutura , Sarcômeros/ultraestrutura , Animais , Imagem Molecular/métodos , Conformação Proteica , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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