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11/25/2008Biochemistry: Motors
Molecular Motors I
Andy HowardIntroductory Biochemistry
25 November 2008
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Chemistry and movement Most purposeful biological motion is effected through actions of molecular motors. It’s worthwhile to understand the biochemistry of these systems
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What we’ll discuss Definition Microtubules and their partners Tubulin Structure Cilia & flagella
Microtubules (concluded) Movement of organelles
Dyneins & kinesins DNA helicases Muscle contraction: for next time!
Bacterial flagella
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What is a molecular motor?
A protein-based system that interconverts chemical energy and mechanical work
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Microtubules
30-nm structures composed of repeating units of a heterodimeric protein, tubulin -tubulin: 55 kDa -tubulin: 55 kDa also
Structure of microtubule itself: polymer in which the heterodimers wrap around in a staggered way to produce a tube
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Tubulin structure
and are similar but not identical Structure determined by electron diffraction, not X-ray diffraction
Some NMR structures available too Two GTP binding sites per monomer Heterodimer is stable if Ca2+ present
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iClicker quiz question 1 Why might you expect crystallization of tubulin to be difficult? (a) It is too big to crystallize (b) It is too small to crystallize (c) Proteins that naturally form complex but non-crystalline 3-D structures are resistant to crystallization
(d) It is membrane-bound (e) none of the above
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Tubulin dimer
G&G Fig. 16.2
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Microtubule structure
Polar structure composed of / dimers
Dimers wrap around tube as they move
Asymmetric: growth at plus end
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Treadmilling Dimers added at plus end while others removed at minus end (GTP-dependent): that effectively moves the microtubule
Fig. 16.3
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Role in cytoskeleton Microtubules have a role apart from their role in molecular motor operations:
They are responsible for much of the rigidity of the cytoskeleton
Cytoskeleton contains: Microtubules (made from tubulin) Intermediate fibers (7-12nm; made from keratins and other proteins)
Microfilaments (8nm diameter: made from actin)
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Cytoskeletal components
Fig. 16.4
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Cilia and flagella Both are microtubule-based structures used in movement
Cilia: short, hairlike projections, found on many animal and lower-plant cells
beating motion moves cells or helps moved extracellular fluid over surface
Flagella Longer, found singly or a few at a time
Propel cells through fluids
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Axonemes
Bundle of microtubule fibers: Two central microtubules Nine pairs of joined microtubules Often described as a 9+2 arrangement
Surrounded by plasma membrane that connects to the cell’s PM
If we remove the PM and add a lot of salt, the axoneme will release a protein called dynein
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Axoneme structure Inner pair connected by bridge
Outer nine pairs connected to each other and to inner pair
Fig. 16.5
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How cilia move
Each outer pair contains asmaller, static A tubule anda larger, dynamic B tubule
Dynein walks along B tubulewhile remaining attached toA tubule of a different pair
Crosslinks mean the axoneme bends Dynein is a complex protein assembly:
ATPase activity in 2-3 dynein heavy chains
Smaller proteins attach at A-tubule end
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Dynein movement Fig. 16.6
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Inhibitors of microtubule polymerization
Vinblastine & vincristine are inhibitors: show antitumor activity by shutting down cell division
Colchicine inhibits microtubule polymerization: relieves gout, probably by slowing movement of white cells
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Paclitaxel: a stimulator
Formerly called taxol Stimulates microtubule polymerization
Antitumor activity Stimulates search for other microtubule polymerization stimulants
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iClicker question 22. How do you imagine paclitaxel might work?
(a) by producing frantic cell division (b) by interfering with microtubule disassembly, preventing cell division
(c ) by causing changes in tertiary structures of and tubulin
(d) none of the above
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Movement of organelles and vacuoles
Can be fast:2-5 µm s-1
Hard to study 1985: Kinesin isolated
1987: Cytosolic dynein found
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Cytosolic dynein
Mostly moves organelles & vesicles from (+) to (-), so it moves things toward the center of the cell
Heavy chain ~ 400kDa, plus smaller peptides (53-74 kDa)
Microtubule-activated ATPase activity
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Kinesin Mostly moves organelles from (-) to (+) That has the effect of moving things outward
360 kDa: 110 kDa heavy chains, also 65-70 kDa subunits (2 + 2?)
Head domain of heavy chain (38 kDa) binds ATP and microtubule: cooperative interactions between pairs of head domains in kinesin, causing conformational changes in a single tubulin subunit
8 nm movements along long axis of microtubule
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Kinesin motion depicted
Rolling movement involving two head domains at a time
Fig. 16.8(b)
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Hand-over-hand kinesin model Two head groups begin in contact
After ATP hydrolysis hindmost head passes forward head
ATP binds to new leading head
Pi dissociates from trailing head
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DNA helicases To replicate DNA we need to separate the strands
Efficient only if the helicase can travel along the duplex quickly
This kind of movement is called processive
E.coli BCD helicase can unwind 33kbp before it falls off
If we want to replicate DNA rapidly, we need processivity
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Achieving processivity
Some helicases form rings that encircle 1 or both strands of the duplex
Others, like rep helicase, are homodimeric; move hand-over-hand along the DNA, like kinesin
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Negative cooperativity Rep is monomeric without DNA Each monomer can bind either ss or dsDNA
BUT after one monomer binds DNA, the second subunit’s affinity drops 104-fold!
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Muscle contraction This is an obvious case of an energy-dependent biological motion system
Involves an interaction called the sliding filament model, in which myosin molecules slide past actin molecules
Many other proteins and structural components involved
We’ll discuss this in detail next Tuesday
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Bacterial flagella E.coli flagellum is 10 µm in length, 15 nm in diameter
~6 filaments on surface of cell rotate counter-clockwise: that makes them bundle together and propel the cell through medium
Enabled by rotation of motor protein complexes in plasma membrane
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Motor structure
>= 2 rings, ~25nm diameter (M & S) Rod attaches those to the helical filament
Rings surrounded by array of membrane proteins
This one is driven by a proton gradient, not by ATP hydrolysis: [H+]out > [H+]in, so protons want to move in
If we let protons in, we can use the thermodynamic energy to drive movement
Requires 800-1200 protons per full rotation!
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The shuttle
MotA & MotB form shuttling device
Proton movement drives rotation of flagellar motor
Fig. 16.26
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iClicker question 3
3. Compare the pH inside the cell to the pH outside.
(a) pHin < pHout
(b) pHin> pHout
(c ) pHin = pHout
(d) We don’t have enough information to answer this question.
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Berg’s model motB has protonexchanging sites
motA has half-channels—one half facing toward the inside of the cell, one facing out
When a motB site is protonated, the outside edges of motA can’t move past it
Center of motA can’t move past site when it’s empty
Those constraints cause coupling between proton translocation and rotation
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Coupling described Proton enters outside of motA and binds
to an exchange site on motB motA is linked to cell wall, so when it rotates, it puts the inside channel over the proton
Proton moves through inside channel into cell; then another proton travels up the outside channel to bind to the next exchange site
That pulls the complex to the left, leading to counterclockwise rotation of disc, rod, & helical filament
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Coupling depicted
Fig. 16.27
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What if it got reversed?
If outside became alkaline, the flagellar filaments would rotate clockwise
That doesn’t work as well because it loosens the microtubule
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Quantitation M ring has about 100 motB exchange sites
800-1200 protons for a full rotation of the filament
That enables ~ 100 rotations/sec
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