Chapter 17. Molecular Motors. to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham. Biochemistry 2/e - Garrett & Grisham

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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

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 Ca 2+ ions from the SR Ca 2+ ions bind to sites on the fibers and induce contraction; relaxation involves pumping the Ca 2+ back into the SR

20 Molecular Structure of Muscle Be able to explain the EM in Figure 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 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 sevenresidue 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 NOT 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 α-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

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 α-dystroglycan - extracellular, binds to merosin (a component of laminin) - mutation in merosin linked to severe congenital muscular dystrophy β-dystroglycan - transmembrane protein that binds dystrophin inside Sarcoglycan complex - α, β, γ - 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 P i 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 Ca 2+ Controls Contraction Ca 2+ Channels and Pumps Release of Ca 2+ from the SR triggers contraction Reuptake of Ca 2+ into SR relaxes muscle So how is calcium released in response to nerve impulses? Answer has come from studies of antagonist molecules that block Ca 2+ 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 Ca 2+ 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 Ca 2+ channel of unusual design Note structure in Figures and Conformation change or Ca 2+ -channel activity of DHP receptor apparently gates the ryanodine receptor, opening and closing Ca 2+ channels Many details are yet to be elucidated!

51 Ca 2+ Regulates Contraction Tropomyosin and troponins mediate the effects of Ca 2+ See Figure In absence of Ca 2+, TnI binds to actin to keep myosin off TnI and TnT interact with tropomyosin to keep tropomyosin away from the groove between adjacent actins But Ca 2+ binding changes all this!

52 Ca 2+ Turns on Contraction Binding of Ca 2+ to TnC increases binding of TnC to TnI, simultaneously decreasing the interaction of TnI with actin This allows tropomyosin to slide down into the actin groove, exposing myosin-binding sites on actin and initiating contraction Since troponin complex interacts only with every 7th actin, the conformational changes must be cooperative

54 Binding of Ca 2+ to Troponin C Four sites for Ca 2+ on TnC - I, II, III and IV Sites I & II are N-terminal; III and IV on C term Sites III and IV usually have Ca 2+ bound Sites I and II are empty in resting state Rise of Ca 2+ levels fills sites I and II Conformation change facilitates binding of TnC to TnI

56 Smooth Muscle Contraction No troponin complex in smooth muscle In smooth muscle, Ca 2+ activates myosin light chain kinase (MLCK) which phosphorylates LC2, the regulatory light chain of myosin Ca 2+ effect is via calmodulin - a cousin of TnC Hormones regulate contraction - epinephrine, a smooth muscle relaxer, activates adenylyl cyclase, making camp, which activates protein kinase, which phosphorylates MLCK, inactivating MLCK and relaxing muscle

59 Smooth Muscle Effectors Useful drugs Epinephrine (as Primatene) is an over-thecounter asthma drug, but it acts on heart as well as on lungs - a possible problem! Albuterol is a more selective smooth muscle relaxer and acts more on lungs than heart Albuterol is used to prevent premature labor Oxytocin (pitocin) stimulates contraction of uterine smooth muscle, inducing labor

Chapter 15. Cytoskeletal Systems. Lectures by Kathleen Fitzpatrick Simon Fraser University Pearson Education, Inc.

Chapter 15. Cytoskeletal Systems. Lectures by Kathleen Fitzpatrick Simon Fraser University Pearson Education, Inc. Chapter 15 Cytoskeletal Systems Lectures by Kathleen Fitzpatrick Simon Fraser University Table 15-1 - Microtubules Table 15-1 - Microfilaments Table 15-1 Intermediate Filaments Table 15-3 Microtubules