Movements of truncated kinesin fragments with a short or an artificial flexible neck

Inoue, Yuichi; Toyoshima, Yoko Y.; Iwane, Atsuko H.; Morimoto, Sachiko; Higuchi, Hideo; Yanagida, Toshio

doi:10.1073/pnas.94.14.7275

Cited by 62 publications

(64 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this way it is possible to rationalize the ¢nding that the direction of ncd heads can be reversed by splicing them to a kinesin tail (Henningsen & Schliwa 1997;Case et al 1997), and the inverse experiment reversing kinesin (Endow & Waligora 1998;Cross 1997b). The conformational change accompanying landing (that corresponding to microtubuleactivated release of trapped ADP) can contribute to the step distance, and this contribution might be ampli¢ed via a lever-arm action, though evidence to date suggests that for ukinesin at least, any such lever must be relatively short (Inoue et al 1997;Romberg et al 1998;Mazumdar et al 1998). It is clear that in the absence of microtubules, both heads trap ADP.…”

Section: Towards a Mechanochemical Modelmentioning

confidence: 99%

The conformational cycle of kinesin

Cross

Crevel

Carter

et al. 2000

Phil. Trans. R. Soc. Lond. B

View full text Add to dashboard Cite

The stepping mechanism of kinesin can be thought of as a programme of conformational changes. We brie£y review protein chemical, electron microscopic and transient kinetic evidence for conformational changes, and working from this evidence, outline a model for the mechanism. In the model, both kinesin heads initially trap Mg¢ADP. Microtubule binding releases ADP from one head only (the trailing head). Subsequent ATP binding and hydrolysis by the trailing head progressively accelerate attachment of the leading head, by positioning it closer to its next site. Once attached, the leading head releases its ADP and exerts a sustained pull on the trailing head. The rate of closure of the molecular gate which traps ADP on the trailing head governs its detachment rate. A speculative but crucial coordinating feature is that this rate is strain sensitive, slowing down under negative strain and accelerating under positive strain.

show abstract

Section: Towards a Mechanochemical Modelmentioning

confidence: 99%

The conformational cycle of kinesin

Cross

Crevel

Carter

et al. 2000

Phil. Trans. R. Soc. Lond. B

View full text Add to dashboard Cite

show abstract

“…To test the sensitivity of our assay, we performed control experiments comparing the torsional behavior of two recombinant molecules: a single-headed, monomeric construct (K351) (16), and a double-headed, dimeric construct (K448) (5), consisting of the first 351 and 448 N-terminal residues of the kinesin heavy chain, respectively (Fig. 1D).…”

Section: Monomeric and Dimeric Kinesin Exhibit Unbounded And Boundedmentioning

confidence: 99%

Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping

Gutiérrez–Medina¹,

Fehr

Block

2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are Ϸ30-fold stiffer than its flexible ''hinge'' regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.microtubule ͉ molecular motor ͉ rotation ͉ single molecule ͉ yaw with one degree accuracy C onventional kinesin (kinesin-1) is a motor protein that transports cargo along microtubules (MTs) in cells, converting the chemical energy of nucleotide hydrolysis into mechanical work (1). The structure of kinesin consists of twin catalytic motor domains (heads) that bind to the MT substrate with nucleotidedependent affinity, joined by neck linkers (NLs) to the N terminus of an extended, ␣-helical coiled-coil (CC) stalk (Fig. 1A). Considerable recent progress has been made in understanding the mechanochemistry of the kinesin motor domains, which enable individual molecules to move processively, undertaking hundreds of successive 8-nm steps while hydrolyzing one ATP molecule per step (2-4). However, little is known about the structure of the stalk or its role in the currently favored model for kinesin movement, the so-called ''asymmetric hand-overhand'' stepping mechanism (5-7). In this mechanism, the heads of kinesin advance in strict alternation, exchanging leading, and trailing roles at each step, analogous to a person walking (1). An asymmetric mechanism requires that the ''left'' and ''right'' steps be slightly different; however, in contrast with a symmetric hand-over-hand walk, it does not require continuous rotation of the kinesin stalk. Consistent with this model, a landmark experiment by the Gelles group (8) that tracked the angular motions of MTs driven by single kinesin motors affixed to a surface did not find evidence for large-scale rotations.Although no net rotations of the kinesin stalk are expected during asymmetric hand-over-hand motion, significant momentary angular changes are nevertheless possible. If kinesin were a nearly rigid molecule, Ϯ180°reorientations of the stalk would be anticipated whenever the two heads exchanged positions during stepping (1, 9). It has been speculated that flexible elements within the kinesin structure may relieve any such momentary torsional strain, facilitating forward motion. Although previous single-molecule studies foun...

show abstract

“…Interest in biological motors is based on both their transduction of energy and their small size and hence their possible relevance to micro͞nanotechnology; the remarkable work of Walker, Vale, Kinosita, Hirokawa, Yanagida and others (9)(10)(11)(12)(13)(14)(15)(16)(17) has transformed our understanding of molecular motors. One outcome has been the design and fabrication of new synthetic motors composed entirely of biological molecules (18,19); another outcome has been the integration of components onto recombinant biological motors (20)(21)(22).…”

mentioning

confidence: 99%

Microoxen: Microorganisms to move microscale loads

Weibel

Garstecki²,

Ryan³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

365

291

View full text Add to dashboard Cite

It is difficult to harness the power generated by biological motors to carry out mechanical work in systems outside the cell. Efforts to capture the mechanical energy of nanomotors ex vivo require in vitro reconstitution of motor proteins and, often, protein engineering. This study presents a method for harnessing the power produced by biological motors that uses intact cells. The unicellular, biflagellated algae Chlamydomonas reinhardtii serve as ''microoxen.'' This method uses surface chemistry to attach loads (1-to 6-m-diameter polystyrene beads) to cells, phototaxis to steer swimming cells, and photochemistry to release loads. These motile microorganisms can transport microscale loads (3-m-diameter beads) at velocities of Ϸ100 -200 m⅐sec ؊1 and over distances as large as 20 cm.biological motors ͉ Chlamydomonas ͉ phototaxis ͉ microfluidics ͉ microspheres T his study demonstrates the biological propulsion of microscale loads by the unicellular photosynthetic algae Chlamydomonas reinhardtii (CR). We exploit the chemistry of the algal cell wall to attach single 1-to 6-m polymer beads to CR. Cells with these ''loads'' attached swim at velocities as high as 100-200 m⅐sec Ϫ1 , approximately the velocity of unmodified cells. CR is phototactic and can be guided by using visible light ( Ϸ 500 nm); we have used this phototaxis to control the transport of microscale loads. A photocleavable linker between the surface of the bead and the cell wall allows us to release loads from the surface of the cell photochemically. We have combined these processes to pick up, transport, guide, and drop off beads by using motile cells.There are many examples of nanometer-scale motors in nature. Within the cell, linear motors, including DNA and RNA polymerase, dyneins, kinesins, and myosin, play a critical role in transcription, mitosis, meiosis, muscle contraction, and transporting organelles and synaptic vesicles (1-5). In eukaryotic mitochondria, a rotary motor, ATP synthase, produces ATP by harnessing the flow of protons down an electrochemical proton gradient (6, 7). Outside of the cell, ciliary dyneins drive the beating of eukaryotic flagella and cilia. In bacteria, a complex of Ϸ20 proteins makes up the remarkable rotary motor that powers the motion of flagella (8).Interest in biological motors is based on both their transduction of energy and their small size and hence their possible relevance to micro͞nanotechnology; the remarkable work of Walker, Vale, Kinosita, Hirokawa, Yanagida and others (9-17) has transformed our understanding of molecular motors. One outcome has been the design and fabrication of new synthetic motors composed entirely of biological molecules (18, 19); another outcome has been the integration of components onto recombinant biological motors (20)(21)(22).Here, we use biological motors intact in cells that use flagella (23). An advantage of this strategy over that using isolated and reconstituted motors is its simplicity. It (i) avoids purification and reconstitution of individual motor proteins, (ii) takes...

show abstract

Movements of truncated kinesin fragments with a short or an artificial flexible neck

Cited by 62 publications

References 28 publications

The conformational cycle of kinesin

The conformational cycle of kinesin

Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping

Microoxen: Microorganisms to move microscale loads

Contact Info

Product

Resources

About