Molecular Motors II;
Muscle Contraction
Andy Howard
Introductory Biochemistry, Fall 2010
15 November 2010
Biochem: Motors 'n Muscles
11/15/2010
Chemistry of muscle
contraction
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The most impressive movement
phenomenon in mesoscopic organisms is
muscle movement. It does have a
biochemical basis, which we’ll explore
today; but first we’ll finish our discussion
of other molecular motor systems
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What we’ll discuss
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Motors, concluded
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Kinesin
DNA Processivity
Flagella
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Skeletal muscle
physiology
Thin filaments: actin,
tropomyosin, troponin
Thick filaments:
myosin
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Biochem: Motors 'n Muscles
Sliding filament model
Dystrophin and
cytoskeletal structure
Coupling of ATP
hydrolysis to
conformational changes
in myosin
Myosin & kinesin
Calcium channels and
troponin C
Smooth muscle
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Kinesin
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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
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Rolling movement involving two head
domains at a time
Fig. 16.8(b)
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Hand-overhand kinesin
model
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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
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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
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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
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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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Bacterial flagella
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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
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>= 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!
… that’s equivalent to ~ 250 ATP’s
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The shuttle
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MotA & MotB form
shuttling device
Proton movement
drives rotation of
flagellar motor
Fig. 16.26
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thought question 3
Compare the pH inside a typical bacterial
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
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motB has proton
exchanging 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
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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
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Fig. 16.27
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What if it got reversed?
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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
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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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Muscle contraction
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This is an obvious case of an energydependent 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
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Essential Question
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How can biological macromolecules,
carrying out conformational changes on
the microscopic, molecular level, achieve
these feats of movement that span the
molecular and macroscopic worlds?
We’ll look at the specifics of muscle
contraction, which is an excellent example
of this phenomenon
Note that Tom Irving, on our faculty, is a
world-recognized expert on muscle
physiology
Prof. Thomas
QuickTime™ and a
decompressor
are needed to see this picture.
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C. Irving
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Skeletal
Muscle
Cell
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T-tubules
enable the
sarcolemmal
membrane to
contact the
ends of the
myofibril
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What are t-tubules and the
sarcoplasmic reticulum for?
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The morphology is all geared to
Ca2+ release and uptake!
Nerve impulses reaching the muscle
produce an "action potential" that
spreads over the sarcolemmal
membrane and into the fiber along
the t-tubule network
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t-tubules and SR, continued
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The signal is passed across the
triad junction and induces release
of Ca2+ ions from the SR
Ca2+ ions bind to sites on the
fibers and induce contraction;
relaxation involves pumping the
Ca2+ back into the SR
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Molecular mechanism of
contraction
Be able to explain the EM in Figure 16.12 (16.2 in
the 4th Ed.) in terms of thin and thick filaments
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Thin filaments are composed of actin polymers
F-actin helix is composed of G-actin monomers
F-actin helix has a pitch of 72 nm
But repeat distance is 36 nm
Actin filaments are decorated with tropomyosin
heterodimers and troponin complexes
Troponin complex consists of: troponin T (TnT),
troponin I (TnI), and troponin C (TnC)
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Myofibrils
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Hexagonal
arrays
shown
(fig. 16.12)
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Actin
monomer
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One
domain
on each
side
(16.13 /
16.3)
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Actin
helices
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Pitch =
72nm
Repeat
= 36
nm
Fig.16.
14
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Thin
filament
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Tropomyosin coiled
coil winds around
the actin helix
Each TM dimer
interacts with 7
actin monomers
Troponin T binds to
TM at head-to-tail
junction
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Composition & Structure of
Thick Filaments
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Myosin - 2 heavy chains, 4 light chains
Heavy chains - 230 kD each
Light chains - 2 pairs of different 20 kD chains
The "heads" of heavy chains have ATPase
activity and hydrolysis here drives contraction
Light chains are homologous to calmodulin
and also to TnC
See structure of heads in Figure 16.16 /
16.5b
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Myosin
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Cartoon
EM
S1 myosin
head
structure
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Repeating Structural Elements
Are the Secret of Myosin’s
Coiled Coils
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7-residue, 28-residue and 196-residue repeats
are responsible for the organization of thick
filaments
Residues 1 and 4 (a and d) of the seven-residue
repeat are hydrophobic; residues 2,3 and 6 (b, c
and f) are ionic
This repeating pattern favors formation of coiled
coil of tails. (With 3.6 - NOT 3.5 - residues per
turn, a-helices will coil!)
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Axial view (fig. 16.17)
Myosin tail: 2-stranded a-helical coiled coil
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More Myosin Repeats!
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28-residue repeat (4 x 7) consists of distinct
patterns of alternating side-chain charge (+
vs -), and these regions pack with regions of
opposite charge on adjacent myosins to
stabilize the filament
196-residue repeat (7 x 28) pattern also
contributes to packing and stability of
filaments
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Myosin packing
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Adjoining molecules offset by ~ 14 nm
Corresponds to 98 residues of coiled coil
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Associated proteins
of Muscle
 a-Actinin, a protein that contains several
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repeat units, forms dimers and contains
actin-binding regions, and is analogous in
some ways to dystrophin
Dystrophin is the protein product of the
first gene to be associated with muscular
dystrophy - actually Duchennes MD
See the box on pages 524-525 / 486-487
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Dystrophin
QuickTime™ and a
decompressor
are needed to see this picture.
New Developments!
Dystrophin is part of a large complex
of glycoproteins that bridges the
inner cytoskeleton (actin filaments)
and the extracellular matrix (via a
protein called laminin)
 Two subcomplexes: dystroglycan
and sarcoglycan
 Defects in these proteins have now
been linked to other forms of
muscular dystrophy
Biochem: Motors 'n Muscles
11/15/2010
Nick Menhart:
BCPS faculty
member
specializing in
dystrophin
research
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Dystrophin, actinin,spectrin
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Characteristic 3-helix regions
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Spectrin-repeat
structure
QuickTime™ and a
decompressor
are needed to see this picture.
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These
characteristic 3helix elements
are found in
actinin, spectrin,
dystrophin
Biochem: Motors 'n Muscles
Spectrin repeat
PDB 1AJ3
NMR
12.8 kDa
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Model for
complex
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Actin-dystrophinglycoprotein
complex
Dystrophin forms
tetramers of
antiparallel
monomers
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The Dystrophin Complex
Links to disease
 a-Dystroglycan - extracellular, binds to
merosin (a component of laminin) mutation in merosin linked to severe
congenital muscular dystrophy
 -Dystroglycan - transmembrane protein
that binds dystrophin inside
 Sarcoglycan complex - a, ,  - all
transmembrane - defects linked to limbgirdle MD and autosomal recessive MD
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Hugh Huxley
The Sliding
Filament Model
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QuickTime™ and a
decompressor
are needed to see this picture.
Many contributors!
Hugh Huxley and Jean Hanson
Andrew Huxley and Ralph Niedergerke
Albert Szent-Györgyi showed that actin
and myosin associate (actomyosin
complex)
Sarcomeres decrease length during
contraction (see Figure 16.19)
Szent-Gyorgyi also showed that ATP
causes the actomyosin complex to
dissociate
Albert Szent-Györgyi
Biochem: Motors 'n Muscles
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decompressor
are needed to see this picture.
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Sliding filaments
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Decrease in sarcomere length happens because
of decreases in width of I band and H zone
No change in width of A band
Thin & thick filaments are sliding past one another
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The Contraction Cycle
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Study Figure 16.20 / 16.9!
Cross-bridge formation is followed by power
stroke with ADP and Pi release
ATP binding causes dissociation of myosin
heads and reorientation of myosin head
Details of the conformational change in the
myosin heads are coming to light!
Evidence now exists for a movement of at
least 35 Å in the conformation change
between the ADP-bound state and ADP-free
state
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Mechanism
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Fig.
16.20
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Actin-myosin
interaction
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QuickTime™ and a
d eco mpres sor
are nee ded to s ee this picture.
Ribbon- and spacefilling representations
Ivan Rayment
QuickTime™ and a
d eco mpres sor
are nee ded to s ee this picture.
Hazel Holden
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Similarities in
Motor Proteins
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Initial events of myosin and
kinesin action are similar
But the conformational changes
that induce movement are
different in myosins, kinesins,
and dyneins
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Myosin &
kinesin
motor
domains
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Relay helix
moves back
and forth like
a piston
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Intramolecular
communication &
conformational
changes
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Myosin and kinesin:
ATP hydrolysis 
conformational change
that gets communicated
to track-binding site
Dynein: not well
understood; involves
AAA ATPases
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Muscle Contraction Is
Regulated by Ca2+
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Ca2+ Channels and Pumps
Release of Ca2+ from the SR triggers
contraction
Reuptake of Ca2+ into SR relaxes muscle
So how is calcium released in response to
nerve impulses?
Answer has come from studies of
antagonist molecules that block Ca2+
channel activity
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Ca2+ triggers
contraction
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Release of Ca2+
through voltage- or
Ca2+-sensitive
channel activates
contraction
Pumps induce
relaxation
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Dihydropyridine Receptor
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In t-tubules of heart and skeletal muscle
Nifedipine and other DHP-like molecules
bind to the "DHP receptor" in t-tubules
In heart, DHP receptor is a voltage-gated
Ca2+ channel
In skeletal muscle, DHP receptor is
apparently a voltage-sensing protein and
probably undergoes voltage-dependent
conformational changes
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Ryanodine Receptor
The "foot structure" in terminal cisternae of
SR
 Foot structure is a Ca2+ channel of unusual
design
 Conformation change or Ca2+ -channel
activity of DHP receptor apparently gates
the ryanodine receptor, opening and
closing Ca2+ channels
 Many details are yet to be elucidated!
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Ryanodine Receptor
QuickTime™ and a
decompressor
are needed to see this picture.

Courtesy
BBRI
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