Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The MukB protein is involved in the process of chromosome partition in Escherichia coli and has a domain structure reminiscent of the eukaryotic motor proteins kinesin and myosin. This has led to the suggestion that MukB may function as a motor protein in vivo. In order to test this idea we have recombinantly expressed the N-terminal domain of MukB (residues 1-342) as a poly-His tagged fusion protein for biochemical characterisation. The purified protein (Muk342) is monomeric and has low basal Mg-ATPase (1.23 min(-1)) and Mg-GTPase (0.17 min(-1)) activities. Muk342 binds with high affinity to the prokaryotic tubulin homologue FtsZ and we have evidence that FtsZ can stimulate nucleotide turnover by Muk342. These properties are consistent with MukB functioning as a motor protein using FtsZ as a track or anchor for generating force within E. coli.
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PMID:Interaction of the N-terminal domain of MukB with the bacterial tubulin homologue FtsZ. 968 55

The surface immobilization methods that allowed single-molecule motility experiments with native kinesin have not worked with the ncd motor protein and other kinesin-related motors. To solve this problem, a surfactant (Pluronic F108) was chemically modified with the metal-chelating group nitrilotriacetic acid (NTA) to allow surface immobilization of histidine-tagged microtubule motors. The chelating surfactant provided a convenient and effective method for immobilization and subsequent motility experiments with a dimeric H-tagged ncd protein (H-N195). In experiments with the absorption of H-N195 to polystyrene (PS) beads coated with F108-NTA, a monolayer of H-N195 bound in the presence of Ni2+, while in the absence of Ni2+, the extent of adsorption of H-N195 to PS beads was greatly reduced. In motility experiments with H-N195 immobilized on F108-NTA-coated surfaces, microtubules moved smoothly and consistently at an average speed of 0.16 +/- 0.01 micrometer/s in the presence of Ni2+, while without Ni2+, no microtubules landed on the F108-NTA-coated surfaces. Investigation of H-N195 motility on the F108-NTA surfaces provided several indications that ncd, unlike kinesin, is not processive. First, a critical H-N195 surface density for microtubule motility of approximately 250 molecules/micrometer(2) was observed. Second, microtubule landing rates as a function of H-N195 surface density in the presence of MgATP suggested that several H-N195 molecules must cooperate in microtubule landing. Third, the ATP KM in motility assays (235 microM) was substantially higher than the ATP KM of dimeric ncd in solution (23 microM) [Foster, K. A., Correia, J. J., and Gilbert, S. P. (1998) J. Biol. Chem. 273, 35307-35318].
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PMID:Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive. 1021 10

A kinesin gene has been cloned by RT-PCR (reverse transcription polymerase chain reaction) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine-tag) kinesin in E. coli in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several liters of culture, an ultrasonic homogenizer was used for cell disruption and an unclarified feedstock was obtained. From this homogenate, a protein was then purified by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology (Streamline chelating). For this capture step, 100% of the recombinant protein was purified with more than 90% of purity. This step was followed by ion-exchange chromatography (Q Sepharose Fast Flow) for intermediate purification (96% purity, 53% recovery) and by size-exclusion chromatography with Superdex 75 as a polishing step (99% purity, 93% recovery). We then separated two forms of kinesin, a dimer (70%) and a monomer (30%). It was then possible to purify His-tag recombinant protein directly from feedstock in a rapid and efficient way and to isolate two forms of kinesin.
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PMID:Three-step chromatographic purification procedure for the production of a his-tag recombinant kinesin overexpressed in E. coli. 1068 Oct 50

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubulebinding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.
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PMID:Two kinesins from Arabidopsis, KatB and KatC, have a second microtubule-binding site in the tail domain. 1724 81

An extensive computational analysis of available sequence and crystal structure data was used to identify functionally important residue interactions within the motor domain of the kinesin molecular motor. Principal component analysis revealed that all current kinesin crystal structures reside in one of two main conformations, which differ at the active site, and in the position of a microtubule-binding sub-domain relative to a rigid central core. This sub-domain consists of secondary structure elements alpha4-loop12-alpha5-loop13 and contains a conserved hydrophilic surface patch that may be involved in strong binding to microtubules. A hinge point for the sub-domain motion lies near a conserved glycine at position 292. Statistical coupling analysis revealed a network of co-evolving positions that link this region to the nucleotide-binding site, via a highly conserved histidine in the switch I loop. The data are consistent with a model in which the nucleotide status of the active site shifts kinesin between weak and strong binding conformations via reconfiguration of the identified sub-domain. Our data provide a statistically supported framework for further examination of this and other structure-function relationships in the kinesin family.
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PMID:Multivariate analysis of conserved sequence-structure relationships in kinesins: coupling of the active site and a tubulin-binding sub-domain. 1739 40

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck-linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck-linker of kinesin by genetically engineering three histidine residues to create a Zn(2+)-binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by >/=10 microM Zn(2+), as well as other divalent metals. Motility was successfully restored by removal of Zn(2+) using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn(2+)/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device.
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PMID:Controlling kinesin motor proteins in nanoengineered systems through a metal-binding on/off switch. 1851 58

Many biological processes require the co-operative involvement of both microtubules and microfilaments; however, only a few proteins mediating the interaction between microtubules and microfilaments have been identified from plants. In the present study, a cotton kinesin GhKCH2, which contains a CH (calponin homology) domain at the N-terminus, was analysed in vitro and in vivo in order to understand its interaction with the two cytoskeletal elements. A specific antibody against GhKCH2 was prepared and used for immunolabelling experiments. Some GhKCH2 spots appeared along a few microtubules and microfilaments in developing cotton fibres. The His-tagged N-terminus of GhKCH2 (termed GhKCH2-N) could co-precipitate with microfilaments and strongly bind to actin filaments at a ratio of monomeric actin/GhKCH2-N of 1:0.6. The full-length GhKCH2 recombinant protein was shown to bind to and cross-link microtubules and microfilaments in vitro. A GFP-fusion protein GFP-GhKCH2 transiently overexpressed in Arabidopsis protoplasts decorated both microtubules and microfilaments, confirming the binding ability and specificities of GhKCH2 on microtubules and microfilaments in living plant cells. The results of the present study demonstrate that GhKCH2, a plant-specific microtubule-dependent motor protein, not only interacts with microtubules, but also strongly binds to microfilaments. The cytoskeletal dual-binding and cross-linking ability of GhKCH2 may be involved in the interaction between microtubules and microfilaments and the biological processes they co-ordinate together in cotton cells.
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PMID:A cotton kinesin GhKCH2 interacts with both microtubules and microfilaments. 1941 90

The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence microscopy of kinesin bound to a carboxylated quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-Delta C) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles.
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PMID:Diffusive movement of processive kinesin-1 on microtubules. 1968 27

In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1-like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1-green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.
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PMID:Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice. 2128 25

To expand the available panel of recombinant proteins that can be useful for identifying Leishmania-infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum, cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4-93.0% and 36.4-97.2%) and specificities (76.1-100% and 90.4-97.3%) when tested with serum samples from Leishmania-infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases.
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PMID:Characterization of novel Leishmania infantum recombinant proteins encoded by genes from five families with distinct capacities for serodiagnosis of canine and human visceral leishmaniasis. 2214 38


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