12/02/2008Biochemistry: Muscles Muscle Contraction Andy Howard Introductory Biochemistry 2 December 2008

12/02/2008Biochemistry: Muscles Page 2 of 46 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

12/02/2008Biochemistry: Muscles Page 3 of 46 What we’ll discuss Skeletal muscle physiology Thin filaments: actin, tropomyosin, troponin Thick filaments: myosin Sliding filament model Dystrophin and cytoskeletal structure Coupling of ATP hydrolysis to conformational changes in myosin Myosin & kinesin Calcium channels and troponin C Smooth muscle

12/02/2008Biochemistry: Muscles Page 4 of 46 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

12/02/2008Biochemistry: Muscles Page 5 of 46 Skeletal Muscle Cell T-tubules enable the sarcolemmal membrane to contact the ends of the myofibril

12/02/2008Biochemistry: Muscles Page 6 of 46 What are t-tubules and SR for? The morphology is all geared to Ca 2+ 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

12/02/2008Biochemistry: Muscles Page 7 of 46 t-tubules and SR, continued 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

12/02/2008Biochemistry: Muscles Page 8 of 46 Molecular mechanism of contraction 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)

12/02/2008Biochemistry: Muscles Page 9 of 46 Myo- fibrils Hexagonal arrays shown (fig )

12/02/2008Biochemistry: Muscles Page 10 of 46 Actin monomer One domain on each side (16.13)

12/02/2008Biochemistry: Muscles Page 11 of 46 Actin helices Pitch = 72nm Repeat = 36 nm Fig

12/02/2008Biochemistry: Muscles Page 12 of 46 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

12/02/2008Biochemistry: Muscles Page 13 of 46 Composition & 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 16.16

12/02/2008Biochemistry: Muscles Page 14 of 46 Myosin Cartoon EM S1 myosin head structure

12/02/2008Biochemistry: Muscles Page 15 of 46 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 NOT residues per turn, a-helices will coil!)

12/02/2008Biochemistry: Muscles Page 16 of 46 Axial view (fig ) Myosin tail: 2-stranded  -helical coiled coil

12/02/2008Biochemistry: Muscles Page 17 of 46 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

12/02/2008Biochemistry: Muscles Page 18 of 46 Myosin packing Adjoining molecules offset by ~ 14 nm Corresponds to 98 residues of coiled coil

12/02/2008Biochemistry: Muscles Page 19 of 46 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

12/02/2008Biochemistry: Muscles Page 20 of 46 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 Nick Menhart: BCPS faculty member specializing in dystrophin research

12/02/2008Biochemistry: Muscles Page 21 of 46 Dystrophin, actinin,spectrin Characteristic 3-helix regions

12/02/2008Biochemistry: Muscles Page 22 of 46 Spectrin-repeat structure These characteristic 3- helix elements are found in actinin, spectrin, dystrophin Spectrin repeat PDB 1AJ3 NMR 12.8 kDa

12/02/2008Biochemistry: Muscles Page 23 of 46 Model for complex Actin-dystrophin- glycoprotein complex Dystrophin forms tetramers of antiparallel monomers

12/02/2008Biochemistry: Muscles Page 24 of 46 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

12/02/2008Biochemistry: Muscles Page 25 of 46 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-Gyorgyi also showed that ATP causes the actomyosin complex to dissociate Hugh Huxley Albert Szent-Györgyi

12/02/2008Biochemistry: Muscles Page 26 of 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

12/02/2008Biochemistry: Muscles Page 27 of 46 The Contraction Cycle Study Figure 16.20! 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 Å in the conformation change between the ADP-bound state and ADP-free state

12/02/2008Biochemistry: Muscles Page 28 of 46 Mechanism Fig

12/02/2008Biochemistry: Muscles Page 29 of 46 Actin-myosin interaction Ribbon- and space- filling representations Ivan Rayment Hazel Holden

12/02/2008Biochemistry: Muscles Page 30 of 46 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

12/02/2008Biochemistry: Muscles Page 31 of 46 Myosin & kinesin motor domains Relay helix moves back and forth like a piston

12/02/2008Biochemistry: Muscles Page 32 of 46 Intramolecular communication & conformational changes Myosin and kinesin: ATP hydrolysis  conformational change that gets communicated to track-binding site Dynein: not well understood; involves AAA ATPases

12/02/2008Biochemistry: Muscles Page 33 of 46 Muscle Contraction Is Regulated by Ca 2+ 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

12/02/2008Biochemistry: Muscles Page 34 of 46 Ca 2+ triggers contraction Release of Ca 2+ through voltage- or Ca 2+ -sensitive channel activates contraction Pumps induce relaxation

12/02/2008Biochemistry: Muscles Page 35 of 46 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

12/02/2008Biochemistry: Muscles Page 36 of 46 Ryanodine Receptor The "foot structure" in terminal cisternae of SR Foot structure is a Ca 2+ channel of unusual design 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!

12/02/2008Biochemistry: Muscles Page 37 of 46 Ryanodine Receptor Courtesy BBRI

12/02/2008Biochemistry: Muscles Page 38 of 46 Muscle Contraction Is Regulated by Ca 2+ 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!

12/02/2008Biochemistry: Muscles Page 39 of 46 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

12/02/2008Biochemistry: Muscles Page 40 of 46 Thin & thick filaments Changes that happen when Ca 2+ binds to troponin C Fig

12/02/2008Biochemistry: Muscles Page 41 of 46 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

12/02/2008Biochemistry: Muscles Page 42 of 46 2 views of troponin C Ribbon Molecular graphic Fig

12/02/2008Biochemistry: Muscles Page 43 of 46 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 Troponin C

12/02/2008Biochemistry: Muscles Page 44 of 46 Effect of hormones on smooth muscle 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

12/02/2008Biochemistry: Muscles Page 45 of 46 Smooth Muscle Effectors Useful drugs Epinephrine (as Primatene) is an over-the- counter 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

12/02/2008Biochemistry: Muscles Page 46 of 46 Oxytocin structure P.532