Motors II; Muscle Contraction

1. Motors II;Muscle Contraction Andy HowardIntroductory Biochemistry1 December 2014 Motors II; Muscle Biochemistry

2. Chemistry of muscle contraction • The most impressive movement phenomenon in mesoscopic organisms is muscle movement. It does have a biochemical basis, which we’ll explore today. • … But first we’ll finish our conversation about other molecular motors Motors II; Muscle Biochemistry

3. Other motors Dynein, Kinesin Helicases Processivity Flagella revisited Skeletal muscle Thin filaments: actin, tropomyosin, troponin Thick filaments: myosin Sliding filament model Dystrophin and cytoskeletal structure Coupling Myosin & kinesin Calcium channels and troponin C, I Smooth muscle What we’ll discuss Motors II; Muscle Biochemistry

4. Movement of organelles and vacuoles • Can be fast:2-5 µm s-1 • Hard to study • 1985: Kinesin isolated • 1987: Cytosolic dynein found Motors II; Muscle Biochemistry

5. Dynein movement Motors II; Muscle Biochemistry

6. 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 Motors II; Muscle Biochemistry

7. 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 Motors II; Muscle Biochemistry

8. Kinesin motion depicted • Rolling movement involving two head domains at a time • Fig. 16.8(b) Motors II; Muscle Biochemistry

9. 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 Motors II; Muscle Biochemistry

10. 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 Motors II; Muscle Biochemistry

11. 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 Motors II; Muscle Biochemistry

12. 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 104-fold! Motors II; Muscle Biochemistry

13. 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 Motors II; Muscle Biochemistry

14. 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!… that’s equivalent to ~ 250 ATP’s Motors II; Muscle Biochemistry

15. The shuttle • MotA & MotB form shuttling device • Proton movement drives rotation of flagellar motor • Fig. 16.26 Motors II; Muscle Biochemistry

16. iClicker question 1 1. Compare the pH inside a typical bacterial cell to the pH outside. • (a) pHin < pHout • (b) pHin> pHout • (c) pHin = pHout • (d) We don’t have enough information to answer this question. Motors II; Muscle Biochemistry

17. Berg’s model • motB has protonexchanging 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 Motors II; Muscle Biochemistry

18. 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 Motors II; Muscle Biochemistry

19. Coupling depicted • Fig. 16.27 Motors II; Muscle Biochemistry

20. 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 Motors II; Muscle Biochemistry

21. Quantitation • M ring has about 100 motB exchange sites • 800-1200 protons for a full rotation of the filament • That enables ~ 100 rotations/sec Motors II; Muscle Biochemistry

22. 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 Motors II; Muscle Biochemistry

23. Essential Question • How can biological macromolecules, carrying out conformational changes on the microscopic, molecular level, achieve these feats of movement that span the molecular and macroscopic worlds? • We’ll look at the specifics of muscle contraction, which is an excellent example of this phenomenon • Note that Tom Irving, on our faculty, is a world-recognized expert on muscle physiology Prof. Thomas C. Irving Motors II; Muscle Biochemistry

24. Skeletal Muscle Cell G&G fig. 16.1 • T-tubules enable the sarcolemmal membrane to contact the ends of the myofibril Motors II; Muscle Biochemistry

25. What are t-tubules (transverse tubules) and the sarcoplasmic reticulum for? • The morphology is all geared to Ca2+ release and uptake! • Nerve impulses reaching the muscle produce an "action potential" that spreads over the sarcolemmal membrane and into the fiber along the t-tubule network Motors II; Muscle Biochemistry

26. t-tubules and SR, continued • The signal is passed across the triad junction and induces release of Ca2+ ions from the SR • Ca2+ ions bind to sites on the fibers and induce contraction; relaxation involves pumping the Ca2+ back into the SR Motors II; Muscle Biochemistry

27. Molecular mechanism of contraction Be able to explain the EM in Figure 16.2in terms of thin and thick filaments • Thin filaments are composed of actin polymers • F-actin helix is composed of G-actin monomers • F-actin helix has a pitch of 72 nm • But repeat distance is 36 nm • Actin filaments are decorated with tropomyosin heterodimers and troponin complexes • Troponin complex consists of: troponin T (TnT), troponin I (TnI), and troponin C (TnC) Motors II; Muscle Biochemistry

28. Myo- fibrils • Hexagonal arrays shown Motors II; Muscle Biochemistry

29. Actin monomer • One domain on each side(fig. 16.3) Motors II; Muscle Biochemistry

30. Actin helices • Pitch = 72nm • Repeat = 36 nm • Cf. Fig.16.4 Motors II; Muscle Biochemistry

31. Thin filament • Tropomyosin coiled coil winds around the actin helix • Each TM dimer interacts with 7 actin monomers • Troponin T binds to TM at head-to-tail junction Motors II; Muscle Biochemistry

32. Composition & Structure of Thick Filaments Myosin - 2 heavy chains, 4 light chains • Heavy chains - 230 kD each • Light chains - 2 pairs of different 20 kD chains • The "heads" of heavy chains have ATPase activity and hydrolysis here drives contraction • Light chains are homologous to calmodulin and also to TnC • See structure of heads in the G&G figure Motors II; Muscle Biochemistry

33. Myosin • Cartoon • EM • S1 myosin head structure • Cf. Fig. 16.5(b) Motors II; Muscle Biochemistry

34. Repeating Structural Elements Are the Secret of Myosin’s Coiled Coils • 7-residue, 28-residue and 196-residue repeats are responsible for the organization of thick filaments • Residues 1 and 4 (a and d) of the seven-residue repeat are hydrophobic; residues 2,3 and 6 (b, c and f) are ionic • This repeating pattern favors formation of coiled coil of tails. (With 3.6 - NOT 3.5 - residues per turn, α-helices will coil!) Motors II; Muscle Biochemistry

35. iClicker question 2 • 2. The lowest-level repeat pattern involves 7 residues; the highest is 196 residues. How many lowest-level repeats are contained in one highest-level repeat? • (a) 196 • (b) 28 • (c) 14 • (d) 7 • (e) undeterminable from the data given. Motors II; Muscle Biochemistry

36. Axial view Myosin tail: 2-stranded -helical coiled coil Motors II; Muscle Biochemistry

37. More Myosin Repeats! • 28-residue repeat (4 x 7) consists of distinct patterns of alternating side-chain charge (+ vs -), and these regions pack with regions of opposite charge on adjacent myosins to stabilize the filament • 196-residue repeat (7 x 28) pattern also contributes to packing and stability of filaments Motors II; Muscle Biochemistry

38. Myosin packing • Adjoining molecules offset by ~ 14 nm • Corresponds to 98 residues of coiled coil Motors II; Muscle Biochemistry

39. Associated proteins of Muscle • -Actinin, a protein that contains several repeat units, forms dimers and contains actin-binding regions, and is analogous in some ways to dystrophin • Dystrophin is the protein product of the first gene to be associated with muscular dystrophy - actually Duchennes MD • See the boxes on this subject in text Motors II; Muscle Biochemistry

40. Dystrophin Dystrophin is part of a large complex of glycoproteins that bridges the inner cytoskeleton (actin filaments) and the extracellular matrix (via a protein called laminin) • Two subcomplexes: dystroglycan and sarcoglycan • Defects in these proteins have now been linked to other forms of muscular dystrophy Nick Menhart:BCPS faculty member specializing in dystrophin research Motors II; Muscle Biochemistry

41. Dystrophin, actinin,spectrin • Characteristic 3-helix regions • Cf. box on pp. 522-523 Motors II; Muscle Biochemistry

42. Spectrin-repeat structure • These characteristic 3-helix elements are found in actinin, spectrin, dystrophin Human dystrophin: first spectrin repeat27 kDa dimer;monomer shownPDB 3UUN, 2.3Å Motors II; Muscle Biochemistry

43. Model for complex • Actin-dystrophin-glycoprotein complex • Dystrophin forms tetramers of antiparallel monomers Motors II; Muscle Biochemistry

44. The Dystrophin Complex Links to disease • -Dystroglycan - extracellular, binds to merosin (a component of laminin) - mutation in merosin linked to severe congenital muscular dystrophy • -Dystroglycan - transmembrane protein that binds dystrophin inside • Sarcoglycan complex - , ,  - all transmembrane - defects linked to limb-girdle MD and autosomal recessive MD Motors II; Muscle Biochemistry

45. Hugh Huxley The Sliding Filament Model Many contributors! • Hugh Huxley and Jean Hanson • Andrew Huxley and Ralph Niedergerke • Albert Szent-Györgyi showed that actin and myosin associate (actomyosin complex) • Sarcomeres decrease length during contraction (see Figure 16.19) • Szent-Györgyi also showed that ATP causes the actomyosin complex to dissociate Albert Szent-Györgyi Motors II; Muscle Biochemistry

46. Sliding filaments • Decrease in sarcomere length happens because of decreases in width of I band and H zone • No change in width of A band • Thin & thick filaments are sliding past one another Motors II; Muscle Biochemistry

47. The Contraction Cycle Study Figure 16.9! • Cross-bridge formation is followed by power stroke with ADP and Pi release • ATP binding causes dissociation of myosin heads and reorientation of myosin head • Details of the conformational change in the myosin heads are coming to light! • Evidence now exists for a movement of at least 35 Å in the conformation change between the ADP-bound state and ADP-free state Motors II; Muscle Biochemistry

48. Mechanism • Fig. 16.9 Motors II; Muscle Biochemistry

49. Actin-myosin interaction • Ribbon- and space-filling representations Ivan Rayment Hazel Holden Motors II; Muscle Biochemistry

50. Similarities in Motor Proteins • Initial events of myosin and kinesin action are similar • But the conformational changes that induce movement are different in myosins, kinesins, and dyneins Motors II; Muscle Biochemistry