Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 126
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-29716949

RESUMO

Myosin motors power movements on actin filaments, whereas dynein and kinesin motors power movements on microtubules. The mechanisms of these motor proteins differ, but, in all cases, ATP hydrolysis and subsequent release of the hydrolysis products drives a cycle of interactions with the track (either an actin filament or a microtubule), resulting in force generation and directed movement.


Assuntos
Dineínas/fisiologia , Cinesina/fisiologia , Miosinas/fisiologia , Citoesqueleto de Actina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico/fisiologia , Dineínas/ultraestrutura , Cinesina/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Miosinas/ultraestrutura
2.
Nat Commun ; 9(1): 1662, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29695795

RESUMO

Kinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promote microtubule depolymerization and lack motile activity. The molecular mechanism by which kinesin-13s depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue, here we report the near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to curved or straight tubulin in different nucleotide states. These structures show how nucleotide induced conformational changes near the catalytic site are coupled with movement of the kinesin-13-specific loop-2 to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.


Assuntos
Proteínas de Drosophila/ultraestrutura , Cinesina/ultraestrutura , Microtúbulos/ultraestrutura , Tubulina (Proteína)/ultraestrutura , Trifosfato de Adenosina/metabolismo , Movimento Celular , Microscopia Crioeletrônica , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Cinesina/química , Cinesina/isolamento & purificação , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Polimerização , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Tubulina (Proteína)/química
3.
Elife ; 62017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28826477

RESUMO

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.


Assuntos
Cinesina/metabolismo , Cinesina/ultraestrutura , Fenômenos Mecânicos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Ligação Proteica , Conformação Proteica
4.
Sci Rep ; 7(1): 6166, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28733669

RESUMO

In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.


Assuntos
Cinesina/ultraestrutura , Microscopia de Força Atômica/métodos , Tubulina (Proteína)/ultraestrutura , Microtúbulos/ultraestrutura , Movimento (Física) , Imagem Individual de Molécula
5.
Elife ; 62017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28504639

RESUMO

The detailed basis of walking by dimeric molecules of kinesin along microtubules has remained unclear, partly because available structural methods have been unable to capture microtubule-bound intermediates of this process. Utilizing novel electron cryomicroscopy methods, we solved structures of microtubule-attached, dimeric kinesin bound to an ATP analog. We find that under these conditions, the kinesin dimer can attach to the microtubule with either one or two motor domains, and we present sub-nanometer resolution reconstructions of both states. The former structure reveals a novel kinesin conformation that revises the current understanding of how ATP binding is coupled to forward stepping of the motor. The latter structure indicates how tension between the two motor domains keeps their cycles out of phase in order to stimulate directional motility. The methods presented here pave the way for future structural studies of a variety of challenging macromolecules that bind to microtubules and other filaments.


Assuntos
Imagem Tridimensional , Cinesina/metabolismo , Cinesina/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Ligação Proteica , Conformação Proteica , Multimerização Proteica
6.
Nat Struct Mol Biol ; 24(5): 461-468, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28394326

RESUMO

Cilia are multifunctional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multimotor assemblies with kinesin-II. Combining these data with insights from single-particle EM, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2's mechanical 'linker' and 'stalk' domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2's recycling activity during IFT.


Assuntos
Dineínas/química , Dineínas/metabolismo , Dineínas/ultraestrutura , Humanos , Cinesina/química , Cinesina/metabolismo , Cinesina/ultraestrutura , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
7.
J Eukaryot Microbiol ; 64(3): 293-307, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27595611

RESUMO

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.


Assuntos
Segregação de Cromossomos , Cilióforos/genética , Cinesina/genética , Cinesina/fisiologia , Meiose , Mitose , Tetrahymena thermophila/genética , Animais , Núcleo Celular , Cilióforos/citologia , Técnicas de Inativação de Genes , Cinesina/classificação , Cinesina/ultraestrutura , Macronúcleo , Prófase Meiótica I , Metáfase , Microtúbulos , Mutação , Filogenia , Proteínas Recombinantes , Fuso Acromático , Polos do Fuso , Tetrahymena/genética , Tetrahymena thermophila/citologia , Tetrahymena thermophila/metabolismo
8.
Proc Natl Acad Sci U S A ; 113(34): 9430-9, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27493215

RESUMO

Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Cinesina/ultraestrutura , Microtúbulos/ultraestrutura , Subunidades Proteicas/química , Tubulina (Proteína)/ultraestrutura , Motivos de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Expressão Gênica , Humanos , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Paclitaxel/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Suínos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
9.
Sci Rep ; 5: 11916, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26132639

RESUMO

Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.


Assuntos
Proteínas Cromossômicas não Histona/ultraestrutura , Cromossomos Humanos/ultraestrutura , Adenosina Trifosfatases/fisiologia , Adenosina Trifosfatases/ultraestrutura , Antígenos de Neoplasias/fisiologia , Antígenos de Neoplasias/ultraestrutura , Proteínas Cromossômicas não Histona/fisiologia , Cromossomos Humanos/fisiologia , DNA Topoisomerases Tipo II/fisiologia , DNA Topoisomerases Tipo II/ultraestrutura , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação a DNA/ultraestrutura , Células HeLa , Humanos , Imagem Tridimensional , Cinesina/fisiologia , Cinesina/ultraestrutura , Metáfase , Complexos Multiproteicos/fisiologia , Complexos Multiproteicos/ultraestrutura
10.
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 Ambiente
11.
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
12.
Biochemistry ; 54(3): 859-69, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25537000

RESUMO

We have employed molecular dynamics (MD) simulation to investigate, with atomic details, the structural dynamics and energetics of three major ATPase states (ADP, APO, and ATP state) of a human kinesin-1 monomer in complex with a tubulin dimer. Starting from a recently solved crystal structure of ATP-like kinesin-tubulin complex by the Knossow lab, we have used flexible fitting of cryo-electron-microscopy maps to construct new structural models of the kinesin-tubulin complex in APO and ATP state, and then conducted extensive MD simulations (total 400 ns for each state), followed by flexibility analysis, principal component analysis, hydrogen bond analysis, and binding free energy analysis. Our modeling and simulation have revealed key nucleotide-dependent changes in the structure and flexibility of the nucleotide-binding pocket (featuring a highly flexible and open switch I in APO state) and the tubulin-binding site, and allosterically coupled motions driving the APO to ATP transition. In addition, our binding free energy analysis has identified a set of key residues involved in kinesin-tubulin binding. On the basis of our simulation, we have attempted to address several outstanding issues in kinesin study, including the possible roles of ß-sheet twist and neck linker docking in regulating nucleotide release and binding, the structural mechanism of ADP release, and possible extension and shortening of α4 helix during the ATPase cycle. This study has provided a comprehensive structural and dynamic picture of kinesin's major ATPase states, and offered promising targets for future mutational and functional studies to investigate the molecular mechanism of kinesin motors.


Assuntos
Adenosina Trifosfatases/metabolismo , Cinesina/química , Cinesina/metabolismo , Simulação de Dinâmica Molecular , Multimerização Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Cinesina/ultraestrutura , Modelos Moleculares , Miosinas/metabolismo , Análise de Componente Principal , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica
13.
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
14.
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
15.
Elife ; 3: e03680, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25209998

RESUMO

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ∼ 7 Šresolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Transporte Biológico , Fenômenos Biomecânicos , Sequência Conservada , Microscopia Crioeletrônica , Humanos , Cinesina/química , Cinesina/ultraestrutura , Cinética , Magnésio/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
Structure ; 22(8): 1173-1183, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25066134

RESUMO

Members of the kinesin-13 subfamily use motor domains in an unconventional fashion to initiate microtubule (MT) depolymerization at MT ends, suggesting unique conformational transitions for lattice engagement, end adaptation, or both. Using hydrogen-deuterium exchange and electron microscopy, we explored conformational changes in free dimeric mitotic centromere-associated kinesin (MCAK) and when bound to a depolymerization intermediate. ATP hydrolysis relaxes the conformation of the dimer, notably in the neck and N-terminal domain. Exchanging ADP in dimeric MCAK with ATP at the MT plus end induces outward curvature in α/ß-tubulin, accompanied by a restructuring of the MCAK neck and N terminus, as it returns to a closed state. Reestablishing a closed dimer induces lateral separation of paired tubulin dimers, which may assist in depolymerization. Thus, full-length ADP-MCAK transitions from an open diffusion-competent configuration to a closed state upon plus end-mediated nucleotide exchange, which is mediated by conformational changes in the N-terminal domains of the dimer.


Assuntos
Cinesina/química , Cinesina/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Animais , Fenômenos Biomecânicos , Linhagem Celular , Cromatografia Líquida , Dimerização , Hidrólise , Cinesina/ultraestrutura , Espectrometria de Massas , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Mariposas , Nefelometria e Turbidimetria , Polimerização , Conformação Proteica
17.
J Biol Chem ; 288(48): 34839-49, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24145034

RESUMO

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.


Assuntos
Adenosina Trifosfatases/metabolismo , Cinesina/química , Microtúbulos/ultraestrutura , Mitose/genética , Adenosina Trifosfatases/química , Adenilil Imidodifosfato/química , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinesina/genética , Cinesina/ultraestrutura , Cinética , Microscopia Eletrônica , Microtúbulos/química , Ligação Proteica/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
18.
Nano Lett ; 13(7): 3434-8, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-23750886

RESUMO

Biomolecular motor systems are attractive for future nanotechnological devices because they can replace nanofluidics by directed transport. However, the lack of methods to externally control motor-driven transport along complex paths limits their range of applications. Based on a thermo-responsive polymer, we developed a novel technique to guide microtubules propelled by kinesin-1 motors on a planar surface. Using electrically heated gold microstructures, the polymers were locally collapsed, creating dynamically switchable tracks that successfully guided microtubule movement.


Assuntos
Cinesina/química , Cinesina/ultraestrutura , Microtúbulos/química , Microtúbulos/ultraestrutura , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Transdutores , Transferência de Energia , Calefação/métodos , Movimento (Física)
19.
PLoS Comput Biol ; 9(2): e1002907, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23459019

RESUMO

Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through "walking" in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A "walking."


Assuntos
Cinesina/química , Cinesina/metabolismo , Simulação de Dinâmica Molecular , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Cinesina/ultraestrutura , Modelos Biológicos
20.
J Am Chem Soc ; 135(12): 4684-7, 2013 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-23477460

RESUMO

A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu(+))/benzophenone (BP) pairs at its periphery (Glue(BP)-FITC) serves as a "photoclickable molecular glue". By multivalent salt-bridge formation between Gu(+) ions and oxyanions, Glue(BP)-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of "optical tweezers".


Assuntos
Benzofenonas/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Guanidina/química , Cinesina/química , Microtúbulos/química , Animais , Cátions Monovalentes/química , Bovinos , Cinesina/ultraestrutura , Microtúbulos/ultraestrutura , Modelos Moleculares , Processos Fotoquímicos , Soroalbumina Bovina/química , Raios Ultravioleta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA