how are biomolecules moved around (within a cell)? single molecule studies of molecular motors where...
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How are biomolecules moved around (within a cell)? Single Molecule Studies of Molecular Motors
Where Physics Meets Biology
Paul R. SelvinPhysics Department,
Biophysics Center
October 12, 2012
How We Move at the Smallest Scale, a (Bio)Physicist’s Perspective
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How do we measure a single molecule
of anything?
Tools of Physics
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What is Fluorescence?
A way to see objects (at low amounts)
In the laboratory, can see a single molecule of fluorescence!
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Cells labeled with fluorophores
HeLa cell
HeLa cell(anaphase)
Neurons
Brainbow(many neurons)
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Green Fluorescent Protein: Genetically-encoded dye
Fluorescent protein from jelly fish
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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.
Diffusion is not sufficient.
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“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)
NervesNeurotransmitters
Sometimes, chemicals need to move a long way… job of molecular motors
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Extra and Intra-cellular MovementLarge and Small scale movement
Muscle
(Myosin,Walking on
Actin)
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What is a molecular motor?
Takes something like gas, converts its chemical energy (burns it)into Mechanical energy (like motion)
Made of molecules—very small, << smaller than a cell
Could call it a molecular engine
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ATP (Adenosine TriPhosphate) is the food (gasoline) for all cells
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.
-- -
-
--Immediate source of energy in the cell
ATP ADP + Pi
+ Energy
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We deal with small distances & forces
Molecular motors move with:Nanometers & picoNewtons
(a billionth of a meter & a trillionth of a Newton)
How do you measure these steps?How big? How much force?
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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 small forces
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We deal with really small distances
Size: Nanometers or even Angstroms
How big are these?Powers of 10 video10- 100um: typical human hair10 um : typical cell size1000 Nanometer = 1 micron
Small distances, small forces
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Cellular Roadways: Microtubules (radial)
Driven in two directions by different “cars”
Microtubules in green and DNA in blue.
Image from http://www.pdn.cam.ac.uk/groups/roperlab/RoperLab/ImageGallery.html
+
-
(kinesin)
(dynein)
It’s congested! Lots of roadblocks, detours
K+
D-
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Kinesin: A Molecular Motor X nm
X nm1 ATP “eaten”
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??)
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16 nm
q655
pixel size is 160nm2 x real time
8.3 nm, 8.3 nm
8.3 nm
16.6 nm
16.6, 0, 16.6 nm, 0…0 nm
16.6 nm
8.3 8.3 nm
Kinesin: Hand-over-hand or Inchworm?
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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
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Super-Accuracy: Nanometer Distances
Fluorescence Imaging with One Nanometer
Accuracy
8 nm steps
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Super-Accuracy: Nanometer Distances
Fluorescence Imaging with One Nanometer
Accuracy
Step size (nm)Time (sec)
8.4 ± 0.7 nm/step
Step size of cargo is 8.4 nm/step
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Kinesin
0 2 4 6 8 10 12 14
0
80
160
240
320
400
480
560
640
720
800
880
960
1040
1120
1200
1280
disp
lace
men
t (nm
)
time(sec)
<step size> = 16.3 nm
y ~ texp(-kt)
Takes 16 nm hand-over-hand steps
16 nm0 nm
16 nm
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Kinesin (HHMI/Harvard)
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Yildiz, Forkey, McKinney, Ha,Goldman and Selvin, Science (2003)
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What about inside the nucleus?
Are molecular motors still hand-over-hand?
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About HCV NS3 helicase1. 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!)
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FRET
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.
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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.
NS3 moving: Inchworm: does a 3-step
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DNA packaging into Viral Envelope
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Mechanism of how DNA is packaged
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Tomishege & Vale, JCB,2000
Thanks for your attention!
The end.