How We Move at the Smallest Scale,
a (Bio)Physicist’s Perspective
Where Physics Meets Biology
How are biomolecules moved around (within a cell)?
Single Molecule Studies of Molecular Motors
Paul R. Selvin
Physics Department,
Biophysics Center
October 12, 2012
How do we measure
a single molecule
of anything?
Tools of Physics
What is Fluorescence?
A way to see objects
(at low amounts)
In the laboratory, can see a single molecule of fluorescence!
Cells labeled with fluorophores
Neurons
HeLa cell
Brainbow
(many neurons)
HeLa cell
(anaphase)
Green Fluorescent Protein:
Genetically-encoded dye
Fluorescent protein
from jelly fish
Biomolecules need to move inside of a Cell
Chromosomes during Cell Division;
Need thousands of molecular motors
(kinesin & dynein)
to move on roadway (microtubules) from A to B.
Neurotransmitters
Nerves
Sometimes, chemicals need to move a
long way… job of molecular motors
“Kinesin can carry a packet of neurotransmitter
from your spine to the tip of your finger in about
two days — a journey that would take a thousand
years if left to simple diffusion.”
(Molloy and Schmitz, Nature, 2005)
Extra and Intra-cellular Movement
Large and Small scale movement
Muscle
(Myosin,
Walking on
Actin)
What is a molecular motor?
Could call it a molecular engine
Takes something like gas,
converts its chemical energy (burns it)
into Mechanical energy (like motion)
Made of molecules—very small, << smaller than a cell
ATP (Adenosine TriPhosphate)
is the food (gasoline) for all cells
Immediate source of energy in the cell
- - -
- -
ATP  ADP + Pi
+
Energy
ATP is “high” energy because three negative charges
are force together
ADP lower energy because one neg. charge is released.
Can use that excess energy to do work.
How do you measure these
steps?How big? How much force?
We deal with small
distances & forces
Molecular motors move with:
Nanometers & picoNewtons
(a billionth of a meter & a trillionth of a Newton)
We deal with small forces
Forces: picoNewtons (pN)
Force on a penny from a flashlight 1
meter away.
Typically;
1 pN (really small) to 60 pN (really big)
We deal with really small distances
Size: Nanometers or even Angstroms
How big are these?
Powers of 10 video
10- 100um: typical human hair
10 um : typical cell size
1000 Nanometer = 1 micron
Cellular Roadways: Microtubules (radial)
Driven in two directions by different “cars”
K+
D-
Image from
http://www.pdn.cam.ac.uk/g
roups/roperlab/RoperLab/I
mageGallery.html
(kinesin)
+
(dynein)
-
Microtubules in green and DNA in blue.
It’s congested! Lots of roadblocks, detours
Kinesin: A Molecular Motor
X nm
1 ATP “eaten”
X nm
All molecular motors rely on “gas”, i.e., “food” for energy.
Produces a certain amount of work.
(e.g., could get 100 mpg; actually get 25 mpg  25% efficient
What is stride length? How much force?
Hand-over-hand vs. Inchworm??)
Kinesin: Hand-over-hand or Inchworm?
q655
8.3 nm, 8.3 nm
16 nm
8.3 nm 8.3 8.3 nm
16.6 nm
16.6 nm
0 nm
16.6, 0, 16.6 nm, 0…
pixel size is 160nm
2 x real time
We borrowed from Hollywood
to solve the step-size and type.
Fluorescence Imaging with
One Nanometer Accuracy
Very good accuracy!
1.5 nm: 100x improvement,
Very quickly! 1-500 msec
Super-Accuracy: Nanometer Distances
Fluorescence Imaging
with One Nanometer
Accuracy
8 nm steps
Quantum
Dot
Kinesin
(CENP-E)
Axoneme
or microtubule
Super-Accuracy: Nanometer Distances
Fluorescence Imaging
with One Nanometer
Accuracy
Kinesin
(CENP-E)
8.4 ± 0.7 nm/step
Count
Quantum
Dot
Position (nm)
Step size of cargo is 8.4 nm/step
Time (sec)
Axoneme
or microtubule
Step size (nm)
Kinesin
100
1200
80
1120
1040
320
288
60
40
960
880
0
-32 -24 -16 -8
800
0
8
16 24 32 40 48 56 64
step size (nm)
720
<step size> = 16.3 nm
60
640
256
224
192
160
128
96
64
32
50
560
0
5.0
5.5
6.0
40
480
count
displacement (nm)
20
displacement (nm)
count
1280
400
320
240
6.5
7.0
7.5
8.0
8.5
time(sec)
30
y ~ texp(-kt)
20
10
160
0
0.0
80
0.2
0.4
0.6
0.8
dwell time (sec)
0
0
16 nm2
16
4 nm
0 nm
6
8
time(sec)
10
12
14
Takes 16 nm hand-over-hand steps
1.0
1.2
9.0
Kinesin
(HHMI/Harvard)
Yildiz, Forkey, McKinney, Ha,
Goldman and Selvin, Science (2003)
What about inside the nucleus?
Are molecular motors still
hand-over-hand?
About HCV NS3 helicase
1. Hepatitis C Virus (HCV) is a deadly virus affecting 170 million
people in the world, but no cure or vaccine. Affects liver.
2. RNA virus.
3. Non-Structural protein 3 (NS3) is needed for viral replication.
4. NS3 unwinds both RNA and DNA duplexes with 3’ overhang
How does NS3 do this?
A molecular motor!
Operates like an inchworm
Step size: 3.4 Å (very small!)
FRET: measure shape changes
(of single biomolecules
Distance dependent interactions between green and red light bulbs
can be used to deduce the shape of the scissors during the function.
NS3 moving: Inchworm: does a 3-step
We made a little movie out of our results and a bit of imagination. The two
domains over the DNA move in inchworm manner, one base at a time per ATP
while the domain below the DNA stays anchored to the DNA. Eventually,
enough tension builds and DNA is unzipped in a three base pairs burst.
DNA packaging into Viral Envelope
Mechanism of how DNA is packaged
Thanks for your attention!
The end.
Tomishege & Vale, JCB,2000