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Cryo-EM Studies of Monomeric and Dim...

Cha, Michael Hyo Keun.

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Cryo-EM Studies of Monomeric and Dimeric Kinesin-Microtubule Complexes.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Cryo-EM Studies of Monomeric and Dimeric Kinesin-Microtubule Complexes./
Author:	Cha, Michael Hyo Keun.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	137 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-10, Section: B.
Contained By:	Dissertations Abstracts International81-10B.
Subject:	Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13863203
ISBN:	9781658491730

Cryo-EM Studies of Monomeric and Dimeric Kinesin-Microtubule Complexes.
Cha, Michael Hyo Keun.

Cryo-EM Studies of Monomeric and Dimeric Kinesin-Microtubule Complexes. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 137 p.

Source: Dissertations Abstracts International, Volume: 81-10, Section: B.

Thesis (Ph.D.)--Yale University, 2019.

This item must not be sold to any third party vendors.

Kinesin proteins are microtubule-based motors that play critical roles in a wide array of processes in the cell. Although the catalytic motor domain is highly conserved amongst kinesins, many homologs exhibit unique structural features that are crucial for its specific functions. Furthermore, the proper functioning of kinesin often depends on oligomeric states such as tetramers (i.e. kinesin-5) that can crosslink two microtubules or dimers (i.e. kinesin-1) that are capable of walking along a single microtubule.The first chapter of my thesis deals with Cin8, a tetrameric kinesin-5 motor in yeast that plays central roles in proper functioning of the mitotic spindle during cell division. Cin8 possesses the unusual ability to switch directionality based on biochemical conditions that modulate motor crowding. I generated a cryo-EM reconstruction of the monomeric Cin8 motor domain bound to a microtubule, which revealed a novel microtubule-binding mode of the class-specific loop 8 (L8). Combined with results from collaborators that showed unexpected ~4:1 superstoichiometric binding per tubulin site, we have proposed a model for how L8-L8 interactions could enable multiple Cin8 interactions per tubulin site to affect motor crowding.The second chapter describes my work on dimeric kinesin-1, which walks processively along microtubules to transport synaptic vesicles in neurons. The structural basis by which dimeric kinesin detaches its trailing head (ADP-bound following Pi release) and propels this 'tethered' head forward to the next binding site (following hydrolysis in the other 'bound' head) remains controversial, and the structural intermediates corresponding to these events have been elusive. Using newly developed cryo-EM methods, we have characterized three different biochemical states representing distinct stages of the kinesin stepping cycle: a 'waiting' state with one empty and one ADP-bound head (200 nM ADP), a catalytic transition state analog (ADP-AlFx) and a 'post-hydrolysis' ADP-Pi state (200 nM ADP + 10 mM Pi). These results fill in two key gaps in the walking mechanism by showing (1) how a forward step may be disfavored prior to ATP binding, and (2) how the coiled-coil stalk domain between kinesin may behave during processive stepping.

ISBN: 9781658491730Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Cryo-electron microscopy

Cryo-EM Studies of Monomeric and Dimeric Kinesin-Microtubule Complexes.
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245 1 0 $a Cryo-EM Studies of Monomeric and Dimeric Kinesin-Microtubule Complexes.
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300 $a 137 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-10, Section: B.
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506 $a This item must not be sold to any third party vendors.
520 $a Kinesin proteins are microtubule-based motors that play critical roles in a wide array of processes in the cell. Although the catalytic motor domain is highly conserved amongst kinesins, many homologs exhibit unique structural features that are crucial for its specific functions. Furthermore, the proper functioning of kinesin often depends on oligomeric states such as tetramers (i.e. kinesin-5) that can crosslink two microtubules or dimers (i.e. kinesin-1) that are capable of walking along a single microtubule.The first chapter of my thesis deals with Cin8, a tetrameric kinesin-5 motor in yeast that plays central roles in proper functioning of the mitotic spindle during cell division. Cin8 possesses the unusual ability to switch directionality based on biochemical conditions that modulate motor crowding. I generated a cryo-EM reconstruction of the monomeric Cin8 motor domain bound to a microtubule, which revealed a novel microtubule-binding mode of the class-specific loop 8 (L8). Combined with results from collaborators that showed unexpected ~4:1 superstoichiometric binding per tubulin site, we have proposed a model for how L8-L8 interactions could enable multiple Cin8 interactions per tubulin site to affect motor crowding.The second chapter describes my work on dimeric kinesin-1, which walks processively along microtubules to transport synaptic vesicles in neurons. The structural basis by which dimeric kinesin detaches its trailing head (ADP-bound following Pi release) and propels this 'tethered' head forward to the next binding site (following hydrolysis in the other 'bound' head) remains controversial, and the structural intermediates corresponding to these events have been elusive. Using newly developed cryo-EM methods, we have characterized three different biochemical states representing distinct stages of the kinesin stepping cycle: a 'waiting' state with one empty and one ADP-bound head (200 nM ADP), a catalytic transition state analog (ADP-AlFx) and a 'post-hydrolysis' ADP-Pi state (200 nM ADP + 10 mM Pi). These results fill in two key gaps in the walking mechanism by showing (1) how a forward step may be disfavored prior to ATP binding, and (2) how the coiled-coil stalk domain between kinesin may behave during processive stepping.
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653 $a Cryo-electron microscopy
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653 $a Microtubule
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710 2 $a Yale University. $b Cell Biology. $3 2101675
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