Molecular Motors I

11/25/2008Biochemistry: Motors

Molecular Motors I

Andy HowardIntroductory Biochemistry

25 November 2008

11/25/2008Biochemistry: Motors Page 2 of 35

Chemistry and movement Most purposeful biological motion is effected through actions of molecular motors. It’s worthwhile to understand the biochemistry of these systems


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


What is a molecular motor?

A protein-based system that interconverts chemical energy and mechanical work


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


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


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


Tubulin dimer

G&G Fig. 16.2


Microtubule structure

Polar structure composed of / dimers

Dimers wrap around tube as they move

Asymmetric: growth at plus end


Treadmilling Dimers added at plus end while others removed at minus end (GTP-dependent): that effectively moves the microtubule

Fig. 16.3


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)


Cytoskeletal components

Fig. 16.4


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


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


Axoneme structure Inner pair connected by bridge

Outer nine pairs connected to each other and to inner pair

Fig. 16.5


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


Dynein movement Fig. 16.6


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


Paclitaxel: a stimulator

Formerly called taxol Stimulates microtubule polymerization

Antitumor activity Stimulates search for other microtubule polymerization stimulants


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


Movement of organelles and vacuoles

Can be fast:2-5 µm s-1

Hard to study 1985: Kinesin isolated

1987: Cytosolic dynein found


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


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


Kinesin motion depicted

Rolling movement involving two head domains at a time

Fig. 16.8(b)


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


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


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


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!


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


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


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!


The shuttle

MotA & MotB form shuttling device

Proton movement drives rotation of flagellar motor

Fig. 16.26


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.


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


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


Coupling depicted

Fig. 16.27


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


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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Molecular Motors I