Exploring Molecular Motors: From Microtubules to Muscle Contraction

1. Chapter 17 Molecular Motors to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

2. Outline • 17.1 Molecular Motors • 17.2 Microtubules and Their Motors • 17.3 Skeletal Muscle Myosin and Muscle Contraction • 17.4 A Proton Gradient Drives the Rotation of Baterial Flagella

3. Tubulin and Microtubules Fundamental components of the eukaryotic cytoskeleton • Microtubules are hollow, cylindrical polymers made from tubulin dimers • 13 tubulin monomers per turn • Dimers add to the "plus" end and dissociate from the "minus" end as in Figure 17.3 • Microtubules are the basic components of the cytoskeleton and of cilia and flagella • Cilia wave; flagella rotate - ATP drives both!

6. Microtubules in Cilia & Flagella • MTs are the fundamental structural unit in cilia and flagella (see axoneme structure, Fig 17.5) • Dynein proteins walk or slide along MTs to cause bending of one MT relative to another • Dynein movement is ATP-driven • See Figures 17.6 and 17.7

10. Microtubules Highways for "molecular motors" • MTs also mediate motion of organelles and vesicles through the cell • In axons, dyneins move organelles + to -, i.e., toward the nucleus • Kinesins move organelles - to + , i.e., away from the nucleus • See Figure 17.8 and compare (a) and (b)

13. Polymerization Inhibitors Therapeutic agents for gout and cancer • Colchicine, from autumn crocus, inhibits MT polymerization, mitosis and also white cell movement - it is a remedy for gout and an inducer of larger, healthier plants • Vinblastine, vincristine also inhibit MT polymerization - anticancer agents • Taxol, from yew tree bark, stimulates polymerization, stabilizes microtubules and inhibits tumor growth, (esp. breast and ovarian)

15. Morphology of Muscle Four types: skeletal, cardiac, smooth and myoepithelial cells • A fiber bundle contains hundreds of myofibrils that run the length of the fiber • Each myofibril is a linear array of sarcomeres • Each sarcomere is capped on ends by a transverse tubule (t-tubule) that is an extension of sarcolemmal membrane • Surfaces of sarcomeres are covered by SR

18. What are t-tubules and SR for?The morphology is all geared to Ca 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

19. What are t-tubules and SR for?The morphology is all geared to Ca release and uptake! • 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

20. Molecular Structure of Muscle Be able to explain the EM in Figure 17.12 in 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)

25. 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 Figure 17.16

27. Repeating Elements in Myosin The secret to ultrastructure • 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, a-helices will coil!)

29. More 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

31. 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 box on pages 548-549

34. Dystrophin New Developments! • 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

37. 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

38. The Sliding Filament Model Many contributors! • Hugh Huxley and Jean Hanson • Andrew Huxley and Ralph Niedergerke • Albert Szent-Gyorgyi showed that actin and myosin associate (actomyosin complex) • Sarcomeres decrease length during contraction (see Figure 17.22) • Szent-Gyorgyi also showed that ATP causes the actomyosin complex to dissociate

41. The Contraction Cycle Study Figure 17.23! • 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 A in the conformation change between the ADP-bound state and ADP-free state

43. Ca2+ Controls Contraction Ca2+ Channels and Pumps • Release of Ca2+ from the SR triggers contraction • Reuptake of Ca2+ into SR relaxes muscle • So how is calcium released in response to nerve impulses? • Answer has come from studies of antagonist molecules that block Ca2+ channel activity

45. Dihydropyridine Receptor In t-tubules of heart and skeletal muscle • Nifedipine and other DHP-like molecules bind to the "DHP receptor" in t-tubules • In heart, DHP receptor is a voltage-gated Ca2+ channel • In skeletal muscle, DHP receptor is apparently a voltage-sensing protein and probably undergoes voltage-dependent conformational changes

48. Ryanodine Receptor The "foot structure" in terminal cisternae of SR • Foot structure is a Ca2+ channel of unusual design • Note structure in Figures 17.27 and 17.28 • Conformation change or Ca2+ -channel activity of DHP receptor apparently gates the ryanodine receptor, opening and closing Ca2+ channels • Many details are yet to be elucidated!