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

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

HeLa cell

HeLa cell(anaphase)

Neurons

Brainbow(many neurons)

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.

Diffusion is not sufficient.

“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

Extra and Intra-cellular MovementLarge and Small scale movement

Muscle

(Myosin,Walking on

Actin)

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

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

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?

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

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

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-

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??)

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?

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

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

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

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 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!)

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.

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

DNA packaging into Viral Envelope

Mechanism of how DNA is packaged

Tomishege & Vale, JCB,2000

Thanks for your attention!

The end.