1. 11/17/2009Biochemistry: Motors Molecular Motors I Andy Howard Introductory Biochemistry 17 November 2009

2. 11/17/2009Biochemistry: Motors Page 2 of 41 Chemistry and movement Most purposeful biological motion is effected through actions of molecular motors. It’s worthwhile to understand the biochemistry of these systems

3. 11/17/2009Biochemistry: Motors Page 3 of 41 What we’ll discuss Leftovers Midterm Definition Microtubules and their partners Tubulin Structure Cilia & flagella Microtubules (concluded) Movement of organelles Dyneins & kinesins DNA helicases Muscle contraction: for next time! Bacterial flagella

4. 11/17/2009Biochemistry: Motors Page 4 of 41 How is sickling related to malaria? Malaria parasite (Plasmodium spp.) infects erythrocytes They’re unable to infect sickled cells So a partially affected cell might survive the infection better than a non-sickled cell Still some argument about all of this Note that most tropical environments have plenty of oxygen around (not a lot of malaria at 2000 meters elevation) Plasmodium falciparum from A.Dove (2001) Nature Medicine 7:389

5. 11/17/2009Biochemistry: Motors Page 5 of 41 Other hemoglobin mutants Because it’s easy to get human blood, dozens of hemoglobin mutants have been characterized Many are asymptomatic Some have moderate to severe effects on oxygen carrying capacity or erythrocyte physiology

6. 11/17/2009Biochemistry: Motors Page 6 of 41 Midterm results Graded A-D, L-Z E-K will be finished this afternoon Graded exams will be recorded right after class, so they can be picked up this afternoon Nonlocal internet exams will be distributed via IIT Online Both answer keys will be posted shortly Generally pretty solid results

7. 11/17/2009Biochemistry: Motors Page 7 of 41 What is a molecular motor? A protein-based system that interconverts chemical energy and mechanical work

8. 11/17/2009Biochemistry: Motors Page 8 of 41 Microtubules 30-nm structures composed of repeating units of a heterodimeric protein, tubulin  -tubulin: 55 kDa  -tubulin: 55 kDa also Structure of microtubule itself: polymer in which the heterodimers wrap around in a staggered way to produce a tube

9. 11/17/2009Biochemistry: Motors Page 9 of 41 Tubulin structure  and  are similar but not identical Structure determined by electron diffraction, not X-ray diffraction Some NMR structures available too Two GTP binding sites per monomer Heterodimer is stable if Ca 2+ present

10. 11/17/2009Biochemistry: Motors Page 10 of 41 iClicker quiz question 1 Why might you expect crystallization of tubulin to be difficult? (a) It is too big to crystallize (b) It is too small to crystallize (c) Proteins that naturally form complex but non-crystalline 3-D structures are resistant to crystallization (d) It is membrane-bound (e) none of the above

11. 11/17/2009Biochemistry: Motors Page 11 of 41 Tubulin dimer G&G Fig. 16.2

12. 11/17/2009Biochemistry: Motors Page 12 of 41 Microtubule structure Polar structure composed of  /  dimers Dimers wrap around tube as they move Asymmetric: growth at plus end

13. 11/17/2009Biochemistry: Motors Page 13 of 41 Treadmilling Dimers added at plus end while others removed at minus end (GTP- dependent): that effectively moves the microtubule Fig. 16.3

14. 11/17/2009Biochemistry: Motors Page 14 of 41 Role in cytoskeleton Microtubules have a role apart from their role in molecular motor operations: They are responsible for much of the rigidity of the cytoskeleton Cytoskeleton contains: Microtubules (made from tubulin) Intermediate fibers (7-12nm; made from keratins and other proteins) Microfilaments (8nm diameter: made from actin)

15. 11/17/2009Biochemistry: Motors Page 15 of 41 Cytoskeletal components Fig. 16.4 /16.11

16. 11/17/2009Biochemistry: Motors Page 16 of 41 Cilia and flagella Both are microtubule-based structures used in movement Cilia: short, hairlike projections, found on many animal and lower-plant cells beating motion moves cells or helps move extracellular fluid over surface Flagella Longer, found singly or a few at a time Propel cells through fluids

17. 11/17/2009Biochemistry: Motors Page 17 of 41 Axonemes Bundle of microtubule fibers: Two central microtubules Nine pairs of joined microtubules Often described as a 9+2 arrangement Surrounded by plasma membrane that connects to the cell’s PM If we remove the PM and add a lot of salt, the axoneme will release a protein called dynein

18. 11/17/2009Biochemistry: Motors Page 18 of 41 Axoneme structure Inner pair connected by bridge Outer nine pairs connected to each other and to inner pair Fig. 16.5

19. 11/17/2009Biochemistry: Motors Page 19 of 41 How cilia move Each outer pair contains a smaller, static A tubule and a larger, dynamic B tubule Dynein walks along B tubule while remaining attached to A tubule of a different pair Crosslinks mean the axoneme bends Dynein is a complex protein assembly: ATPase activity in 2-3 dynein heavy chains Smaller proteins attach at A-tubule end

20. 11/17/2009Biochemistry: Motors Page 20 of 41 Dynein movement Fig. 16.6

21. 11/17/2009Biochemistry: Motors Page 21 of 41 Inhibitors of microtubule polymerization Vinblastine & vincristine are inhibitors: show antitumor activity by shutting down cell division Colchicine inhibits microtubule polymerization: relieves gout, probably by slowing movement of white cells

22. 11/17/2009Biochemistry: Motors Page 22 of 41 Paclitaxel: a stimulator Formerly called taxol Stimulates microtubule polymerization Antitumor activity Stimulates search for other microtubule polymerization stimulants

23. 11/17/2009Biochemistry: Motors Page 23 of 41 iClicker question 2 2. How do you imagine paclitaxel might work? (a) by producing frantic cell division (b) by interfering with microtubule disassembly, preventing cell division (c ) by causing changes in tertiary structures of  and  tubulin (d) none of the above

24. 11/17/2009Biochemistry: Motors Page 24 of 41 Movement of organelles and vacuoles Can be fast: 2-5 µm s -1 Hard to study 1985: Kinesin isolated 1987: Cytosolic dynein found

25. 11/17/2009Biochemistry: Motors Page 25 of 41 Cytosolic dynein Mostly moves organelles & vesicles from (+) to (-), so it moves things toward the center of the cell Heavy chain ~ 400kDa, plus smaller peptides (53-74 kDa) Microtubule-activated ATPase activity

26. 11/17/2009Biochemistry: Motors Page 26 of 41 Kinesin Mostly moves organelles from (-) to (+) That has the effect of moving things outward 360 kDa: 110 kDa heavy chains, also 65-70 kDa subunits (2 + 2?) Head domain of heavy chain (38 kDa) binds ATP and microtubule: cooperative interactions between pairs of head domains in kinesin, causing conformational changes in a single tubulin subunit 8 nm movements along long axis of microtubule

27. 11/17/2009Biochemistry: Motors Page 27 of 41 Kinesin motion depicted Rolling movement involving two head domains at a time Fig. 16.8(b)

28. 11/17/2009Biochemistry: Motors Page 28 of 41 Hand-over- hand kinesin model Two head groups begin in contact After ATP hydrolysis hindmost head passes forward head ATP binds to new leading head Pi dissociates from trailing head

29. 11/17/2009Biochemistry: Motors Page 29 of 41 DNA helicases To replicate DNA we need to separate the strands Efficient only if the helicase can travel along the duplex quickly This kind of movement is called processive E.coli BCD helicase can unwind 33kbp before it falls off If we want to replicate DNA rapidly, we need processivity

30. 11/17/2009Biochemistry: Motors Page 30 of 41 Achieving processivity Some helicases form rings that encircle 1 or both strands of the duplex Others, like rep helicase, are homodimeric; move hand-over-hand along the DNA, like kinesin

31. 11/17/2009Biochemistry: Motors Page 31 of 41 Negative cooperativity Rep is monomeric without DNA Each monomer can bind either ss or dsDNA BUT after one monomer binds DNA, the second subunit’s affinity drops 10 4 -fold!

32. 11/17/2009Biochemistry: Motors Page 32 of 41 Muscle contraction This is an obvious case of an energy- dependent biological motion system Involves an interaction called the sliding filament model, in which myosin molecules slide past actin molecules Many other proteins and structural components involved We’ll discuss this in detail Thursday

33. 11/17/2009Biochemistry: Motors Page 33 of 41 Bacterial flagella E.coli flagellum is 10 µm in length, 15 nm in diameter ~6 filaments on surface of cell rotate counter-clockwise: that makes them bundle together and propel the cell through medium Enabled by rotation of motor protein complexes in plasma membrane

34. 11/17/2009Biochemistry: Motors Page 34 of 41 Motor structure >= 2 rings, ~25nm diameter (M & S) Rod attaches those to the helical filament Rings surrounded by array of membrane proteins This one is driven by a proton gradient, not by ATP hydrolysis: [H + ] out > [H + ] in, so protons want to move in If we let protons in, we can use the thermodynamic energy to drive movement Requires 800-1200 protons per full rotation!

35. 11/17/2009Biochemistry: Motors Page 35 of 41 The shuttle MotA & MotB form shuttling device Proton movement drives rotation of flagellar motor Fig. 16.26

36. 11/17/2009Biochemistry: Motors Page 36 of 41 iClicker question 3 3. Compare the pH inside a typical bacterial cell to the pH outside. (a) pH in < pH out (b) pH in > pH out (c ) pH in = pH out (d) We don’t have enough information to answer this question.

37. 11/17/2009Biochemistry: Motors Page 37 of 41 Berg’s model motB has proton exchanging sites motA has half-channels—one half facing toward the inside of the cell, one facing out When a motB site is protonated, the outside edges of motA can’t move past it Center of motA can’t move past site when it’s empty Those constraints cause coupling between proton translocation and rotation

38. 11/17/2009Biochemistry: Motors Page 38 of 41 Coupling described Proton enters outside of motA and binds to an exchange site on motB motA is linked to cell wall, so when it rotates, it puts the inside channel over the proton Proton moves through inside channel into cell; then another proton travels up the outside channel to bind to the next exchange site That pulls the complex to the left, leading to counterclockwise rotation of disc, rod, & helical filament

39. 11/17/2009Biochemistry: Motors Page 39 of 41 Coupling depicted Fig. 16.27

40. 11/17/2009Biochemistry: Motors Page 40 of 41 What if it got reversed? If outside became alkaline, the flagellar filaments would rotate clockwise That doesn’t work as well because it loosens the microtubule

41. 11/17/2009Biochemistry: Motors Page 41 of 41 Quantitation M ring has about 100 motB exchange sites 800-1200 protons for a full rotation of the filament That enables ~ 100 rotations/sec