1. How are biomolecules moved around (within a cell)? Single Molecule Studies of Molecular Motors Where Physics Meets Biology Paul R. Selvin Physics Department, Biophysics Center October 12, 2012 How We Move at the Smallest Scale, a (Bio)Physicist’s Perspective

2. How do we measure a single molecule of anything? Tools of Physics

3. What is Fluorescence? A way to see objects (at low amounts) In the laboratory, can see a single molecule of fluorescence!

4. Cells labeled with fluorophores HeLa cell (anaphase) Neurons Brainbow (many neurons)

5. Green Fluorescent Protein: Genetically-encoded dye Fluorescent protein from jelly fish

6. 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.

7. “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) Nerves Neurotransmitters Sometimes, chemicals need to move a long way… job of molecular motors

8. Extra and Intra-cellular Movement Large and Small scale movement Muscle (Myosin, Walking on Actin)

9. 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

10. 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 + P i + Energy

11. 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?

12. 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

13. 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 Small distances, small forces

14. 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/g roups/roperlab/RoperLab/I mageGallery.html http://www.pdn.cam.ac.uk/g roups/roperlab/RoperLab/I mageGallery.html + - (kinesin) (dynein) It’s congested! Lots of roadblocks, detours K+ D-

15. Kinesin: A Molecular Motor X nm 1 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. 16 nm q655 pixel size is 160nm 2 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 8.38.3 nm Kinesin: Hand-over-hand or Inchworm?

17. 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

18. Super-Accuracy: Nanometer Distances Fluorescence Imaging with One Nanometer Accuracy Quantum Dot Kinesin (CENP-E) Axoneme or microtubule 8 nm steps

19. Super-Accuracy: Nanometer Distances Fluorescence Imaging with One Nanometer Accuracy Quantum Dot Kinesin (CENP-E) Axoneme or microtubule Step size (nm) Count Time (sec) Position (nm) 8.4 ± 0.7 nm/step Step size of cargo is 8.4 nm/step

20. Kinesin = 16.3 nm y ~ texp(-kt) Takes 16 nm hand-over-hand steps 16 nm 0 nm 16 nm

21. Kinesin (HHMI/Harvard)

22. Yildiz, Forkey, McKinney, Ha, Goldman and Selvin, Science (2003)

23. What about inside the nucleus? Are molecular motors still hand-over-hand?

24. 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!)

25. 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.

26. 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

27. DNA packaging into Viral Envelope

28. Mechanism of how DNA is packaged

29. Tomishege & Vale, JCB,2000 Thanks for your attention! The end.