Motor Proteins and Cell Motility

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• Motor Proteins and Cell Motility

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Myosin
Dynein
Kinesin
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Molecular motion is powered by the interaction
between molecular motors and their tracts
Actin is the track that myosin interacts with to
produce motion Actin monomers (G) polymerize
into (F) filaments Polymerization is driven by
ATP binding, in the presence of Mg and salt.
Following ATP hydrolysis, ADP remains bound to
the active site of F-actin. F-Actin arranged in a
two strand helix with asymmetric ends called
end and end Growth of the polymer occurs faster
toward the end.
Minus
Plus
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Actin Polymerization
Nucleation is rate limiting step After a period
of fast filament growth filament reaches a
steady-state. Critical concentration monomers
left in solution at steady state. KonC
Koff Koff/Kon 1/K Cc Cells use catalysts of
nucleation to control filament formation and cell
shape
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Polymerization based Motility
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Tethered Ratchet Model
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Elastic Propulsion Model
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Myosin
Myosin from muscle tissue is a 230 kDa protein
that associated with two light-chains (20 kDa)
and forms dimer. Each myosin consists of globular
head and a tail that forms a coiled coil for
dimerization 20 different myosin genes identified
in the human genome many uncharacterized
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Proteolytic Fragments of Myosin
Full length myosin insoluable at low ionic
strength
HMM two headed dimer S1 Motor - crystal
structure solved ATP , Actin, Light Chain
binding
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Decorated Actin Filaments
Pointed end
Arrowhead pattern determines polarity of actin
filament
Barbed end
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Myosin Filament Formation
Bipolar Structure of myosin filaments
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Organization of Skeletal Muscle
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Structure of the Sarcomere


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Sliding Filament Hypothesis
Interdigitated thick and thin filaments slide
past one another to shorten sarcomere Myosin
heads drive shortening by pulling on the actin
filament Using energy of ATP hydrolysis
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Myosin ATPase Cycle
Strong-binding
Weak-binding
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Rotating Cross-Bridge Model of Myosin
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Muscle Movie
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Myosin Structure
Motor Domain
Actin-Binding Region
RLC
Active Site
ELC
Actin-Binding Cleft
Lever Arm
Rayment et al., 1993
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G Proteins and Myosin
P-loop Switch I Switch II
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Common Structural Core of Motor Proteins and
G-Proteins
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Actomyosin Structure
Motor Domain
Lever Arm
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Structure/Function of Myosin Motor
Actin-Binding Cleft
Active Site
Lever Arm
Swinging Lever Arm Generates Force Actin-Binding
Cleft Mediates Actin Binding Active Site
Nucleotide Coordination and Communication
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Myosin 6
-Pointed-end directed myosin motor -Moves
processively -Takes 25 nm steps with a single IQ
domain
Cramer et al., JCB, 2000
Rock et al., PNAS, 2001
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Myosin is a molecular motor
Microneedle Assay myosin generates pN forces
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Myosin is the molecular motor that drives muscle
contraction
In Vitro Motility Assay
Kron and Spudich, 1986
Correlate Biophysical Properties with Biochemical
Properties
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Myosin II vs. Myosin V
Muscle Contraction
Muscle many myosin molecules interacting with
actin to generate force Low Duty Ratio
Organelle Transport
Myosin V single molecule of myosin V walks
along actin to transport organelles High Duty
Ratio
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Duty Ratio
?on average time attached to filament working
stroke ?off average time detached from filament
recovery stroke kATPase Maximum rate of ATP
hydrolysis ?total 1/KATPase V rate of
filament movement r 1/Nmin Velocity KATPase x
?
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Single Molecule Biophysics of Actomyosin
Interactions
Displacement 5-10 nm per myosin
Finer and Spudich, 1996
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Thin filament Regulatory Proteins
Tropomyosin - spans 7 actin monomers joins end
to end Troponin 3 subunits TnC Calcium
binding TnT- binds to tropomyosin TnI inhibits
myosin binding
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Calcium triggers muscle contraction
Calcium binds to TnC and a structural change is
transmitted to move TnI and Tropomyosin away from
the myosin binding site on actin Cooperative
myosin binding can move Tm out of the way
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Calcium Release from Sarcoplasmic Reticulum
A nerve impulse travels along the transverse
tubules Depolarization of membrane triggers
calcium release from the SR through voltage
gated calcium channels
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Many motile processes involving myosin motors
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Cytokinesis
Myosin and actin are found in many cell
types Functions Cell division
cytokinesis Organelle transport Cell shape Cell
motility Exocytosis/Phagocytosis Sensory
transduction
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Contractile Mechanism in Non-muscle Cells
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Structure of Dynein
AAA Family of Proteins six subdomains forming a
ring structure (in dynein they are fused together
into a single polypepetide) One subdomain
contains MT activated ATPase Stalk MT binding
domain Two heavy chains joined together at the
rod-like stem Dynactin complex binds to the stalk
to coordinate cargo binding Displacement along
the MT conformation change in ring (compact to
open moves stalk)
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Intracellular Transport
Kinesin, Cytoplasmic Dynein participate in long
range intracellular transport (highways) Kinesi
n structure similar to myosin more
compact Kinesin moves toward to plus end of
microtubules. Some forms can move toward minus
end Dynein moves toward the minus end Myosin
moves along actin for shorter distances (side
roads)
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Kinesin Movie
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Enzymatic Cycle of Kinesin
Processive Walking occurs by hand-over-hand
mechanism Coordination between two heads
enzymatic steps are effected by mechanical strain
on the motor ATP binding cause conf. change in
kinesin rotates trailing head to allow it to
bind to next site on MT One head bound at all
times weak-binding (ADP), strong binding (ATP
and nucleotide-free)
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Microtubules and Mitosis
Mitotic Spindle microtubule depolymerization/pol
ymerization drives the separation of chromosomes
from the spindle pole Polar MT extend between
centrioles, and help separate them Kinetochore MT
connected to the kinetochores pull toward poles
during telophase
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Steps and Forces

• Working distance (?) distance that cross-bridge
  moves during attached phase of ATPase cycle
• Distance per ATP (?) distance each motor domain
  moves during the time it takes to complete one
  ATPase cycle
• ? Speed of movement (V) / ATPase rate per head
  
• Path distance (d) distance between consecutive
  binding sites on the path that the crossbridge
  follows
• Structural basis of duty ratio
• n?d
• When n 0. There is a futile ATPase cycle with
  no displacement high load
• When ngt1. The motor jumps over one or more
  stepping stones.
• Duty ratio (r) working distance (?) / distance
  per ATP(?)
• r ?/n?d