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1.
Int J Mol Sci ; 20(19)2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31623357

RESUMO

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.


Assuntos
Trifosfato de Adenosina/metabolismo , Cinesina/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Trifosfato de Adenosina/química , Algoritmos , Fenômenos Químicos , Ativação Enzimática , Hidrólise , Cinesina/química , Fenômenos Mecânicos , Proteínas Motores Moleculares/química
2.
Elife ; 82019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31313986

RESUMO

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator-rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator-rotor interaction at an unprecedented detail. Importantly, the stator-rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.


Assuntos
Proteínas de Bactérias/química , Borrelia burgdorferi/química , Flagelos/química , Proteínas Motores Moleculares/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Tomografia com Microscopia Eletrônica , Flagelos/genética , Flagelos/fisiologia , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/fisiologia , Mutação/genética , Rotação , Deleção de Sequência , Sódio/química , Torque
3.
Phys Rev E ; 99(4-1): 042405, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31108695

RESUMO

We consider an explicit model of a semiflexible filament moving in two dimensions on a gliding assay of motor proteins, which attach to and detach from filament segments stochastically, with a detachment rate that depends on the local load experienced. Attached motor proteins move along the filament to one of its ends with a velocity that varies nonlinearly with the motor protein extension. The resultant force on the filament drives it out of equilibrium. The distance from equilibrium is reflected in the end-to-end distribution, modified bending stiffness, and a transition to spiral morphology of the polymer. The local stress dependence of activity results in correlated fluctuations in the speed and direction of the center of mass leading to a series of ballistic-diffusive crossovers in its dynamics.


Assuntos
Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Difusão , Fenômenos Mecânicos
4.
Phys Biol ; 16(4): 046004, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31013252

RESUMO

Active networks composed of filaments and motor proteins can self-organize into a variety of architectures. Computer simulations in two or three spatial dimensions and including or omitting steric interactions between filaments can be used to model active networks. Here we examine how these modelling choices affect the state space of network self-organization. We compare the networks generated by different models of a system of dynamic microtubules and microtubule-crosslinking motors. We find that a thin 3D model that includes steric interactions between filaments is the most versatile, capturing a variety of network states observed in recent experiments. In contrast, 2D models either with or without steric interactions which prohibit microtubule crossings can produce some, but not all, observed network states. Our results provide guidelines for the most appropriate choice of model for the study of different network types and elucidate mechanisms of active network organization.


Assuntos
Microtúbulos/química , Proteínas Motores Moleculares/química , Simulação por Computador , Reagentes para Ligações Cruzadas/química , Citoesqueleto/metabolismo , Multimerização Proteica , Transdução de Sinais
5.
Proc Natl Acad Sci U S A ; 116(13): 5902-5907, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850521

RESUMO

How does a biomolecular machine achieve high speed at high efficiency? We explore optimization principles using a simple two-state dynamical model. With this model, we establish physical principles-such as the optimal way to distribute free-energy changes and barriers across the machine cycle-and connect them to biological mechanisms. We find that a machine can achieve high speed without sacrificing efficiency by varying its conformational free energy to directly link the downhill, chemical energy to the uphill, mechanical work and by splitting a large work step into more numerous, smaller substeps. Experimental evidence suggests that these mechanisms are commonly used by biomolecular machines. This model is useful for exploring questions of evolution and optimization in molecular machines.


Assuntos
Proteínas Motores Moleculares/síntese química , Metabolismo Energético , Transferência de Energia , Modelos Teóricos , Conformação Molecular , Proteínas Motores Moleculares/química , Estrutura Molecular
6.
Molecules ; 24(2)2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646587

RESUMO

Kinesin-1, kinesin-2 and kinesin-5 are three families of a superfamily of motor proteins; which can walk processively on microtubule filaments by hydrolyzing ATP. It was experimentally shown that while the three kinesin dimers show similar feature on the force dependence of velocity, they show rather different features on the force dependence of run length. However, why the three families of kinesins show these rather different features is unclear. Here, we computationally studied the movement dynamics of the three dimers based on our proposed model. The simulated results reproduce well the available experimental data on the force dependence of velocity and run length. Moreover, the simulated results on the velocity and run length for the three dimers with altered neck linker lengths are also in quantitative agreement with the available experimental data. The studies indicate that the three families of kinesins show much similar movement mechanism and the rather different features on the force dependence of run length arise mainly from the difference in rate constants of the ATPase activity and neck linker docking. Additionally, the asymmetric (limping) movement dynamics of the three families of homodimers with and without altered neck linker lengths are studied, providing predicted results.


Assuntos
Cinesina/química , Modelos Teóricos , Proteínas Motores Moleculares/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Cinesina/genética , Proteínas Motores Moleculares/genética , Família Multigênica , Multimerização Proteica
7.
Hamostaseologie ; 39(1): 87-94, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29996171

RESUMO

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). Patients present congenital macrothrombocytopenia and inclusions of NMMHC-IIA in leukocytes, and have a variable risk of developing kidney damage, sensorineural deafness, presenile cataracts and/or liver enzymes abnormalities. The spectrum of mutations found in MYH9-RD patients is limited and the incidence and severity of the non-congenital features are predicted by the causative MYH9 variant. In particular, different alterations of the C-terminal tail domain of NMMHC-IIA associate with remarkably different disease evolution. We report four novel MYH9 mutations affecting the tail domain of NMMHC-IIA and responsible for MYH9-RD in four families. Two variants cause amino acid substitutions in the coiled-coil region of NMMHC-IIA, while the other two are a splicing variant and a single nucleotide deletion both resulting in frameshift alterations of the short non-helical tailpiece. Characterization of phenotypes of affected individuals shows that all of these novel variants are associated with a mild clinical evolution of the disease.


Assuntos
Transtornos Cromossômicos/genética , Proteínas Motores Moleculares/genética , Mutação , Cadeias Pesadas de Miosina/genética , Trombocitopenia/congênito , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Quebra Cromossômica , Transtornos Cromossômicos/patologia , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Motores Moleculares/química , Cadeias Pesadas de Miosina/química , Linhagem , Fenótipo , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Trombocitopenia/genética , Trombocitopenia/patologia , Adulto Jovem
8.
Org Biomol Chem ; 17(1): 53-65, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30534753

RESUMO

Linear motor proteins including kinesin and myosin are promising biomaterials for developing nano-devices. Photoswitchable substrates of these biomotors can be used to optically regulate the motility of their associated cytoskeletal filaments in in vitro systems. Here, we describe the discovery of the myosin selective azobenzene-tethered triphosphate. It enables the specific photocontrol over myosin in a reversible mode with the composite motility assay composed of both kinesin and myosin. The mechanistic insight into this myosin selectivity is also explained with the docking simulation study.


Assuntos
Cinesina/química , Proteínas Motores Moleculares/química , Miosinas/química , Processos Fotoquímicos , Polifosfatos/química , Compostos Azo , Ensaios de Migração Celular , Simulação de Acoplamento Molecular , Especificidade por Substrato
9.
Acc Chem Res ; 51(12): 3015-3022, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30376292

RESUMO

Biomolecular motors, such as the motor protein kinesin, can be used as off-the-shelf components to power hybrid nanosystems. These hybrid systems combine elements from the biological and synthetic toolbox of the nanoengineer and can be used to explore the applications and design principles of active nanosystems. Efforts to advance nanoscale engineering benefit greatly from biological and biophysical research into the operating principles of motor proteins and their biological roles. In return, the process of creating in vitro systems outside of the context of biology can lead to an improved understanding of the physical constraints creating the fitness landscape explored by evolution. However, our main focus is a holistic understanding of the engineering principles applying to systems integrating molecular motors in general. To advance this goal, we and other researchers have designed biomolecular motor-powered nanodevices, which sense, compute, and actuate. In addition to demonstrating that biological solutions can be mimicked in vitro, these devices often demonstrate new paradigms without parallels in current technology. Long-term trends in technology toward the deployment of ever smaller and more numerous motors and computers give us confidence that our work will become increasingly relevant. Here, our discussion aims to step back and look at the big picture. From our perspective, energy efficiency is a key and underappreciated metric in the design of synthetic motors. On the basis of an analogy to ecological principles, we submit that practical molecular motors have to have energy conversion efficiencies of more than 10%, a threshold only exceeded by motor proteins. We also believe that motor and system lifetime is a critical metric and an important topic of investigation. Related questions are if future molecular motors, by necessity, will resemble biomolecular motors in their softness and fragility and have to conform to the "universal performance characteristics of motors", linking the maximum force and mass of any motor, identified by Marden and Allen. The utilization of molecular motors for computing devices emphasizes the interesting relationship among the conversion of energy, extraction of work, and production of information. Our recent work touches upon these topics and discusses molecular clocks as well as a Landauer limit for robotics. What is on the horizon? Just as photovoltaics took advantage of progress in semiconductor fabrication to become commercially viable over a century, one can envision that engineers working with biomolecular motors leverage progress in biotechnology and drug development to create the engines of the future. However, the future source of energy is going to be electricity rather than fossil or biological fuels, a fact that has to be accounted for in our future efforts. In summary, we are convinced that past, ongoing, and future efforts to engineer with biomolecular motors are providing exciting demonstrations and fundamental insights as well as opportunities to wander freely across the borders of engineering, biology, and chemistry.


Assuntos
Bioengenharia , Modelos Biológicos , Dineínas/química , Dineínas/metabolismo , Cinesina/química , Cinesina/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo
10.
PLoS Comput Biol ; 14(9): e1006344, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30222728

RESUMO

Filamentous actin (F-actin) and non-muscle myosin II motors drive cell motility and cell shape changes that guide large scale tissue movements during embryonic morphogenesis. To gain a better understanding of the role of actomyosin in vivo, we have developed a two-dimensional (2D) computational model to study emergent phenomena of dynamic unbranched actomyosin arrays in the cell cortex. These phenomena include actomyosin punctuated contractions, or "actin asters" that form within quiescent F-actin networks. Punctuated contractions involve both formation of high intensity aster-like structures and disassembly of those same structures. Our 2D model allows us to explore the kinematics of filament polarity sorting, segregation of motors, and morphology of F-actin arrays that emerge as the model structure and biophysical properties are varied. Our model demonstrates the complex, emergent feedback between filament reorganization and motor transport that generate as well as disassemble actin asters. Since intracellular actomyosin dynamics are thought to be controlled by localization of scaffold proteins that bind F-actin or their myosin motors we also apply our 2D model to recapitulate in vitro studies that have revealed complex patterns of actomyosin that assemble from patterning filaments and motor complexes with microcontact printing. Although we use a minimal representation of filament, motor, and cross-linker biophysics, our model establishes a framework for investigating the role of other actin binding proteins, how they might alter actomyosin dynamics, and makes predictions that can be tested experimentally within live cells as well as within in vitro models.


Assuntos
Actinas/química , Actomiosina/química , Citoesqueleto de Actina/química , Trifosfato de Adenosina/química , Animais , Fenômenos Biomecânicos , Movimento Celular , Simulação por Computador , Reagentes para Ligações Cruzadas/química , Citoplasma/química , Drosophila , Hidrólise , Proteínas dos Microfilamentos/química , Proteínas Motores Moleculares/química , Contração Muscular , Miosinas/química , Polímeros , Viscosidade , Xenopus laevis
12.
Biochem Biophys Res Commun ; 504(4): 709-714, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30213631

RESUMO

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.


Assuntos
Proteínas de Bactérias/química , Proteínas Motores Moleculares/química , ATPases Translocadoras de Prótons/química , Imagem Individual de Molécula/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Polarização de Fluorescência , Cinesina/química , Cinesina/metabolismo , Cinética , Microscopia de Fluorescência , Proteínas Motores Moleculares/metabolismo , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo , Rotação
13.
FEBS J ; 285(18): 3402-3421, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30066435

RESUMO

Type IVa pili are bacterial appendages involved in diverse physiological processes, including electron transfer in Geobacter sulfurreducens. ATP hydrolysis coupled with conformational changes powers the extension (PilB) and retraction (PilT) motors in the pilus machinery. We report the unliganded crystal structures of the core ATPase domain of PilB and PilT-4 from G. sulfurreducens at 3.1 and 2.6 Å resolution, respectively. PilB structure revealed three distinct conformations, that is, open, closed, and open' which were previously proposed to be mediated by ATP/ADP binding. PilT-4 subunits, on the other hand, were observed in the closed state conformation. We further report that both PilB and PilT-4 hexamers have two high-affinity ATP-binding sites. Comparative structural analysis and solution data presented here supports the "symmetric rotary model" for these ATPase motors. Our data further suggest that pores of these motors rotate either clockwise or counterclockwise to facilitate assembly or disassembly of right-handed or left-handed pilus. DATABASE: Structural data are available in the RCSB PDB database under the PDB ID 5ZFQ (PilT-4), 5ZFR (PilB).


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/fisiologia , Geobacter/enzimologia , Proteínas Motores Moleculares/química , Oxirredutases/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Conformação Proteica , Homologia de Sequência
15.
Methods Mol Biol ; 1805: 151-184, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971718

RESUMO

Mechanical transitions in molecular motors often occur on a submillisecond time scale and rapidly follow binding of the motor with its cytoskeletal filament. Interactions of nonprocessive molecular motors with their filament can be brief and last for few milliseconds or fraction of milliseconds. The investigation of such rapid events and their load dependence requires specialized single-molecule tools. Ultrafast force-clamp spectroscopy is a constant-force optical tweezers technique that allows probing such rapid mechanical transitions and submillisecond kinetics of biomolecular interactions, which can be particularly valuable for the study of nonprocessive motors, single heads of processive motors, or stepping dynamics of processive motors. Here we describe a step-by-step protocol for the application of ultrafast force-clamp spectroscopy to myosin motors. We give indications on optimizing the optical tweezers setup, biological constructs, and data analysis to reach a temporal resolution of few tens of microseconds combined with subnanometer spatial resolution. The protocol can be easily generalized to other families of motor proteins.


Assuntos
Proteínas Motores Moleculares/química , Pinças Ópticas , Actinas/metabolismo , Animais , Avidina/metabolismo , Biotinilação , Calibragem , Bovinos , Análise de Dados , Elasticidade , Corantes Fluorescentes/química , Camundongos , Microesferas , Miosina Tipo II/química , Miosina Tipo V/química , Polimerização , Dióxido de Silício/química
16.
J Mol Biol ; 430(22): 4557-4579, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29959924

RESUMO

Over the past 50 years, protein complexes have been studied with techniques such as X-ray crystallography and electron microscopy, generating images which although detailed are static and homogeneous. More recently, limited application of in vivo fluorescence and other techniques has revealed that many complexes previously thought stable and compositionally uniform are dynamically variable, continually exchanging components with a freely circulating pool of "spares." Here, we consider the purpose and prevalence of protein exchange, first reviewing the ongoing story of exchange in the bacterial flagella motor, before surveying reports of exchange in complexes across all domains of life, together highlighting great diversity in timescales and functions. Finally, we put this in the context of high-throughput proteomic studies which hint that exchange might be the norm, rather than an exception.


Assuntos
Bactérias/metabolismo , Flagelos/metabolismo , Proteínas Motores Moleculares/metabolismo , Bactérias/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fluorescência , Proteínas Motores Moleculares/química , Proteômica
17.
Acta Crystallogr D Struct Biol ; 74(Pt 6): 585-594, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29872008

RESUMO

In situ structural information on molecular machines can be invaluable in understanding their assembly, mechanism and evolution. Here, the use of electron cryotomography (ECT) to obtain significant insights into how an archetypal molecular machine, the bacterial flagellar motor, functions and how it has evolved is described. Over the last decade, studies using a high-throughput, medium-resolution ECT approach combined with genetics, phylogenetic reconstruction and phenotypic analysis have revealed surprising structural diversity in flagellar motors. Variations in the size and the number of torque-generating proteins in the motor visualized for the first time using ECT has shown that these variations have enabled bacteria to adapt their swimming torque to the environment. Much of the structural diversity can be explained in terms of scaffold structures that facilitate the incorporation of additional motor proteins, and more recent studies have begun to infer evolutionary pathways to higher torque-producing motors. This review seeks to highlight how the emerging power of ECT has enabled the inference of ancestral states from various bacterial species towards understanding how, and `why', flagellar motors have evolved from an ancestral motor to a diversity of variants with adapted or modified functions.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Evolução Molecular , Flagelos/química , Proteínas Motores Moleculares/química , Proteínas de Bactérias/química , Flagelos/ultraestrutura
18.
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 , Imagem Tridimensional , Modelos Biológicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/ultraestrutura , Montagem de Vírus
19.
Bioconjug Chem ; 29(7): 2278-2286, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29932650

RESUMO

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.


Assuntos
Citoesqueleto/química , Microtúbulos/química , Proteínas Motores Moleculares/química , Pontos Quânticos/química , Animais , Cromatografia de Afinidade , Complexo Dinactina/isolamento & purificação , Dineínas/isolamento & purificação , Humanos , Proteínas Motores Moleculares/isolamento & purificação , Coloração e Rotulagem , Fatores de Tempo
20.
J Biomol NMR ; 71(4): 237-245, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29948439

RESUMO

Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein. We uniformly 13C/15N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural "hotspots" for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin.


Assuntos
Marcação por Isótopo/métodos , Proteínas Motores Moleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Bactérias/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , DnaB Helicases/química , Helicobacter pylori/química , Isótopos de Nitrogênio , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos
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