Lecture

16 : Linear Molecular Motors

Dr Eileen Nugent

Processive Motion of Motor Protein

Processive = continuous motion of
single molecule Organelles being carried
along by kinesin and dyenin
kinesin typically travels ~ 10 m before motors
detaching (~ 1000 steps)

How Proteins Work : Garland Science

 One-State Ratchet Models

More detailed models of Linear Motors:

Intermediate States, Two-State Ratchets

Polymerization Ratchets

One-state ratchets
Cartoon picture of e.g
kinesin moving
on a microtubule, 4 nm
periodic protein structure

Linear motors move along regular, periodic tracks

One-state model : forward stepping with force dependent rate constant k+

backward stepping with rate constant k-
all bound motors equivalent

Free energy difference between steps:

 Motor Stepping
Real free energy isnt flat between steps
but more like this:

In principle we could solve the

Smolunchowski eqn for this landscape; in
practice we dont know its details

Rate limiting step is diffusion over a

free energy barrier
L
Transition rates can be related to barrier
height using Arrhenius relation

Kinesin Stepping on a Microtubule

Kinesin stepping data : makes discrete steps, sometimes backwards

Ref : Carter and Ross, Nature 435:308 (2005)

 Myosin Stepping on Actin
Myosin stepping on actin mainly forwards but some backwards stepping

Muscle myosin : Myosin V : Transport molecule

Not very processive Highly processive

Refs : Kitamura et al, Biophysics 1:119. (2005)

Mehta et al, Nature 400:590 (1999)

V([ATP]) predictions
Two parameters : forwards and backwards stepping rate constants related by
Thermodynamic arguments

Either can change with [ATP] but v([ATP]) different depending on which one

All dependence in
backward rate

Speed saturates with [ATP]

All dependence in
forward rate

Speed is linear with [ATP]

 Kinesin : v([ATP])
Kinesin data not consistent with pure k- or k+ modulation
Kinesin not a simple one state ratchet

Saturates at high [ATP]

Linear at low [ATP]

One-state ratchets
Dwell time distributions
How do we know that cleverer forms for k([ATP]) and k+([ATP]) wouldnt fit the data for
[ATP])?
If transitions between states are controlled by activation barriers, the waiting time for each transition will be
exponentially distributed (with mean time " = 1/k)
One-state and two-state waiting time distributions look fundamentally different:

t/
For transitions controlled by activation
For a one-state ratchet we expect a dwell time distribution p(t) e

barriers waiting time for transition

Exponentially distributed with mean time
A two-state ratchet moves when two sequential transitions (each a simple
exponential) have occurred. The total dwell time distribution should look like p(t) e
t/ 1
e t/ 2

Can distinguish between one-state rel. number of event

1.0

0.8

0.6

and two-state waiting time distributions 0.4

0.2

time
0 2 4 6 8 10
 Experimental Evidence

<- Step data from individual kinesin

molecules stepping in microtubules

From which a distribution of

Waiting times can be obtained |
V

Kinesin has a bi-exponential dwell time

distribution: its at least a two-state
ratchet

Myosin V also at least two-state (not

shown here)

One-State Ratchet Models

More detailed models of Linear Motors:

Intermediate States, Two-State Ratchets

Polymerization Ratchets
 One-State Model Assumptions
Two-state ratchets
Most motor free energy landscapes cannot be reduced to a single
Most motor energy landscapes cannot be reduced to a single (distance)
distance reaction coordinate
reaction coordinate.
We Single transition state assumption not accurate
oversimplified by assuming there was a single transition state: WRONG.

Work (motion against f) and

burning of ATP occur concurrently
transition state

one-state model one-state cartoon

Additional States
Two-state ratchets
More detailed model with intermediate states required
One more reaction coordinate
Decouple ATP hydrolysis from physical movement
Make a more detailed model with intermediate 856
state(s)
CHAPTER 16. DYNAMICS OF MOLECULAR MOTORS
Complete decoupling ATP hydrolysis insensitive to load f, physical movement insensitive
This adds (at least) another reaction coordinate
to [ATP] This allows ATP burning (potentially 3 steps in itself) to be decoupled from physical movement.
If they are completely decoupled, the former will be insensitive to f and the latter will be insensitive to [ATP].

(A)
+ +
kB kA

kB kA

1 0 1

transition state(s)

n1 n n+1

+ +
(B) kA kB

kA kB

0 1 0

transition state(s)

n1 n n+1

Figure 16.32: Two-state motor model. (A) The rates for the transitions that
multi-state model two-state cartoon
can occur to change the occupancy of internal state 0. The dark icon indicates
the current state of the motor head and the two light icons indicate the two
possible states in the next time step. In the two-state model, the motor head is
constrained to convert from internal state 0 to internal state 1. This can occur
either while the motor remains stationary with respect to the filament (with
rate constant kA ) or while the motor takes a single step backwards (with rate
constant kB ) (B) The rates for the transitions that can occur to change the
occupancy of internal state 1. The motor head can convert to internal state 0,
either while remaining in place, or while taking a single step forward.
 Two State Motor Models

Consider motor as two state

system with states 0,1
Find p0(n,t) and p1(n,t) protein
being in state 0 on subunit n at time t
(similarly state 1)
K values give rates of transitions to
different internal or position states

Rate equations:

Can reduce this

to probability of being KAs change state and n
in state 0 or 1 irrespective KBs change state
of location n

Parameterized problem
for one site is enough to
capture dynamics

Assume : 0 -> 1
while remaining on same
site gives movement
0->1 moving to neighbouring
site gives movement a
(a subunit size kinesin 8 nm)
Myosin V and Two-State Models
Two-state ratchets
Orthogonal Energy release and motion looks reasonably good
For myosin V, the two-state model with orthogonal energy release and motion looks good:
Bi-exponential fitting of dwell time distributions gives :
Fitting the dwell time distributions to biexponentials gives one rate constant (k1) that is completely independent of
- k1 completely independent of [ATP]
[ATP] and one rate constant (k2) that is almost independent of load.
Unfortunately, PBoC chooses to display a dwell time distribution that looks monoexponential because k ! k1 at very high [ATP]
- k2 almost independent of load
2

PBoC also switches k and k compared to the source paper.

2 1

Fig. 4. Force and ATP dependence for MV-1IQ-S1. Rates shown are derived
from a maximum-likelihood fit of the dwells to two sequential rates. Rates
from different ATP concentrations are plotted on a log-log scale. The fits are
estimates based on one ATP-independent rate (k1) and one saturating ATP-
dependent rate (k2).
Fig. 4. Force and ATP dependence for MV-1IQ-S1. Rates shown are derived
from a maximum-likelihood fit of the dwells to two sequential rates. Rates
Purcell et al, A force-dependent from
state controls the coordination of on a log-log scale. The fits are
myosin Vdifferent
to actin.ATP concentrations
This are plotted
load dependence suggests that this
processive myosin V, PNAS 102:1387313878
estimates
transition based
involves a mechanical(2005)
on one ATP-independent
displacement.rate
This(k1) and oneissaturating ATP-
transition
dependent
hypothesized to rate
occur(k2).
while the actin-bound motor is in its ADP
Ref : Purcell et al, PNAS 102:1387313878 (2005)
and actin-bound state, changing from its weakly actin-bound
(A!M!ADP) to its strongly actin-bound (A!M*!ADP) state. A
myosin
backward loadVleads
to to
actin. This in
an increase load dependence
the motors suggests
population of that this
weakly bound state,
transition resulting
involves in the motorsdisplacement.
a mechanical dissociation fromThis
the transition is
actinhypothesized
filament at "1.5 s !1. Recent studies on unloaded single-
to occur while the actin-bound motor is in its ADP
and actin-bound state, changing from its weakly actin-bound
(A!M!ADP) to its strongly actin-bound (A!M*!ADP) state. A
backward load leads to an increase in the motors population of
weakly bound state, resulting in the motors dissociation from the
actin filament at "1.5 s!1. Recent studies on unloaded single-

Kinesin Walking : Closer detail

Fig. 3. Dwell-time histograms for MV-6IQ-S1. Data are shown for both
pulling forward (F, mimicking a trailing head) or pulling backward (!, mim-
icking a leading head). Data are fit as two sequential rates fk1,k2(t) # {k1
k2!(k1 ! k2)} $ (e!k2t ! e!k1t) or fit as a single exponential rate f(t) # k e!kt.
(A) At 1,000 !M ATP, the forward pulling is fit to a single rate of 15 s!1 (n #
532). The backward-pulling distribution fits to a single rate of 1.5 s!1 (n # 398).
(B) At 10 !M ATP, the forward pulling is fit to two rates (n # 1,071), and the
backward pulling is fit to a single rate (n # 1,222). The dashed line is a fit to
two rates, 1.7 and 24 s!1. The deviation shows that any other steps other than
1.6-s!1 observed
Fig. 3. the Dwell-time off rate must
histograms be %%24 s!1. (C)
for MV-6IQ-S1. At 1,000
Data !M ATP,
are shown theboth
for
pullingforward
forward pulling is fit to a single rate of 3.7 s!1 (n # 662) and the backward
(F, mimicking a trailing head) or pulling backward (!, mim-
pulling is fit to 1.3 s!1 (n # 553). (D) Dwell-time histogram for MV-4IQ-S1 at 10
icking !aMleading
ATP.
head). Data are fit as two sequential rates fk1,k2(t) # {k1
k2!(k1 ! k2)} $ (e!k2t ! e!k1t) or fit as a single exponential rate f(t) # k e!kt.
(A) At 1,000 !M ATP, the forward pulling is fit to a single rate of 15 s!1 (n #
532). The backward-pulling distribution fits to!1
the assay. The rates at 11 and 15 s a single rate of 1.5 s!1 (n # 398).
are within error of the
(B) At 10 !M ATP, the forward pulling is fit to two rates (n # 1,071), and the
established ADP release rate. Data from MV-1IQ-S1 are con-
backward pulling is fit to a single rate (n # 1,222). The dashed line is a fit to
sistent with a second-order rate constant for ATP-induced actin
two rates, 1.7 and 24 s!1. The deviation shows that any other steps other thanFig. 5. Off-axis strain accelerates ADP release. Cartoon representation of
unbinding of "2 !M!1!s!1. In addition, the rates for pulling MV-4IQ-HMM attached to a segment of actin. The lead head is shown bound
the 1.6-s!1 observed off rate must be %%24 s!1. (C) At 1,000 !M ATP, the
forward are similar regardless of the !1 length of the lever arm.
Scripps Institute
at the second, sixth, seventh, and 11th actin subunit, corresponding to a span
forward pulling is fit to a single rate of 3.7 s (n # 662) and the backwardof 5.5, 16.5, 19.3, and 30.3 nm between the two heads. The intermediate
Pulling backward on MV-6IQ-S1 reduces the apparent dwell
pulling is fit to 1.3 s (n # 553). (D) Dwell-time histogram for MV-4IQ-S1 at 10distances require the myosin to twist around the actin helix, as shown by the
!1
time to a single measurable rate. Under this condition, the dwell
!M ATP. arrows indicating the orientation of each myosin head. Processive steps from
time distribution is not affected by the concentration of ADP or
MV-4IQ-HMM at 10 !M ATP were collected into 4-nm bins, and the times for
ATP. This finding suggests that the unbinding occurs before the the preceding and following dwells were fit to two rates. Closed symbols show
ADP release step (Fig. 6). We hypothesize that a backward load the two rates that best-fit prestep dwells, and open symbols show the rates
the assay. The rates
is inhibiting at 11 and 15
the weak-binding s are withintransition
to strong-binding
!1
error ofof thederived from poststep dwells.
established ADP release rate. Data from MV-1IQ-S1 are con-
sistent13876
with" awww.pnas.org!cgi!doi!10.1073!pnas.0506441102
second-order rate constant for ATP-induced actin Purcell et al.
Fig. 5. Off-axis strain accelerates ADP release. Cartoon representation of
unbinding of "2 !M!1!s!1. In addition, the rates for pulling MV-4IQ-HMM attached to a segment of actin. The lead head is shown bound
forward are similar regardless of the length of the lever arm. at the second, sixth, seventh, and 11th actin subunit, corresponding to a span
Pulling backward on MV-6IQ-S1 reduces the apparent dwell of 5.5, 16.5, 19.3, and 30.3 nm between the two heads. The intermediate
time to a single measurable rate. Under this condition, the dwell distances require the myosin to twist around the actin helix, as shown by the
arrows indicating the orientation of each myosin head. Processive steps from
time distribution is not affected by the concentration of ADP or
MV-4IQ-HMM at 10 !M ATP were collected into 4-nm bins, and the times for
ATP. This finding suggests that the unbinding occurs before the the preceding and following dwells were fit to two rates. Closed symbols show
ADP release step (Fig. 6). We hypothesize that a backward load the two rates that best-fit prestep dwells, and open symbols show the rates
is inhibiting the weak-binding to strong-binding transition of derived from poststep dwells.

13876 " www.pnas.org!cgi!doi!10.1073!pnas.0506441102 Purcell et al.

 Kinesin
Tightly coupled (one spatial
step for each ATP
molecule consumed)

Highly processive
(many steps before
detachment)

Nearly 100% duty cycle

A tightly-coupled molecular motor with at least one irreversible step in kinetics should move
at a speed according to MM kinetics (with parameters dependent on the load force)

Kinesin Structure and Biochemistry

 Forward step
against the load

Models of Kinesin
There is still considerable disagreement about how kinesin works.

Two competing models:

A powerstoke model, where ATP binding and/or hydrolysis causes a conformational
change, amplified through a lever arm, that moves kinesin

A diffusion-to-capture model, where ATP binding / ADP release control head attachment
to microtubule but motion occurs by passive diffusion of one head past the other.

There are also hybrid models where ADP release causes a conformation change
(twisting of the linker) that biases diffusion .

The main problem of both models is how to coordinate ATP processing in the
two kinesin heads. Assumption is that strain transferred from the leading head
to the trailing (through the linker) is responsible.
 Motion without tight coupling

Bind at n

Unbind and
diffuse freely

Rebind at
n-1,n, or n+1

1 vs 2 headed Kinesin
 One-State Ratchet Models

More detailed models of Linear Motors:

Intermediate States, Two-State Ratchets

Polymerization Ratchets

Polymerization Ratchet
Addition of monomers generates a force
When filament reaches the barrier (e.g. cell wall) fluctuations
in position of cell wall or filament will allow another monomer
to squeeze through and bind
 Polymerization Ratchet contd

How do we find the velocity (filament

polymerization rate)?
Driven diffusion equation (F-P)
Want to find p(x) of finding a particle
at x in width
Force p(x) to be 1 in (0,) interval
Mean rate at which particle reaches
boundary at x = starting at x = 0 is

We want to solve for p(x) in terms of

j0

Velocity of Polymerization Ratchet

The solution is the sum of the general solution of homogeneous
Equation (absence of force) and a particular solution to inhomogeneous equation:

Low Force: Same as no force limit diffusion first passage problem

High Force:
Probability particle
will find itself with
energy F