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1、Chapter 5-4Chapter 5-4 Protein function, modulation and evolution: Myosin and actinMyosin being the prototype of a molecular motor, converting chemical energy of ATP into mechanical energy by cyclical interaction with the actin filament, generating force and movement. Key in muscle contraction; cell
2、 division; organelle streaming, etc.Biochemistry Lecture for Oct 16, 2012EMstudiesBiochemical studiesProtein-based molecular motors allow organisms to “move”at various levelslMany things are moving in living organisms, e.g.:Muscle contraction in vertebrates (myosin and actin);Migration of organelles
3、 along microtubules and chromosome separation in dividing cells (kinesins and dyneins);Rotation of bacterial flagella (a complex rotational motor proteins);DNA metabolism (helicases and polymerases).lVia cyclic conformational changes of proteins, consuming chemical energy supplied by ATP.lAchieving
4、exceptionally high levels of spatial and temporal organization.Sarcomere make the structural and functional unit of myofibrilsZ lineZ lineStriated appearance: Alternating light and dark bands;I-band: Zone of thin filaments (of actin) alone; Z-line: the middle of thin filaments;A-band: Entire length
5、of a single thick filament (of myosin);M-line: the middle cross-linked region of thick filaments;H zone: Zone of thick filaments alone; (The A band does not change, but I band and H zone shortensduring muscle contraction!) AnisotropicisotropicLeeuwenhoek first discovered such muscle cross-striations
6、 in 1682! The striated muscle sarcomere is made up of thick (myosin) and thin (actin) filaments, and other proteinsproteins fromskeletal muscle; & smooth muscle (c)Electron micrograph of a longitudinal section of skeletal muscle fiber.Cross sectionsMyosin and actin are two major protein components o
7、f striated muscleG-actinG-actinF-actinF-actinMyosinMyosin: : 520 kDa (two heavy520 kDa (two heavychain, each of 220 kDa; fourchain, each of 220 kDa; fourLight chains, each 20kDa)Light chains, each 20kDa)ActinActin: 42 kDa: 42 kDaCoiled coilsCoiled coilsATPaseATPasebinds actinbinds actinCycle stops h
8、ere in relaxed living muscle (due to removal of myoplasmic calcium)Ca2+*High actin affinityADP+PiADP+PiPiADP*Low actin affinity*ATPATPResting musclePower stroke(ATP bound(ATP boundform of myosin)form of myosin)(ADP bound(ADP boundform of myosin)form of myosin)Muscle contraction model:Muscle contract
9、ion model:Power stroke caused by Power stroke caused by cross-bridge formation,cross-bridge formation,tilting and sliding.tilting and sliding.MyosinMyosinActinActinCross-bridgeCross-bridgeTilting and slidingTilting and slidingMyosin was initially described by Wilhelm F. Kuhne (1864)lA viscous protei
10、n was extracted from the press juice of the frog skeletal muscle with concentrated salt solution.lIt was considered to be responsible for the rigor of muscle.lHe coined the terms “myosin” (also “enzyme”).KuhneKuhne(1837-1900)(1837-1900)Myosin was found to be extremely asymmetric (1930)lMuralt & Edsa
11、ll (1930) Studies in the physical chemistry of muscle globulin, IV. The anisotropy of myosin and double refraction of flow, J. Biol. Chem. 89:351386.lAs indicated by the observations of double refraction of flow and viscosity.lBoth acid and alkali (denaturization) destroyed the double refraction.Myo
12、sin was found to be an ATPaselEngelhardt WA, Liubimova MN. (1939) Myosine and adenosinetriphosphatase, Nature, 144:688-189 lAcidification to pH below 4 rapidly destroys this activity; completely lost after 10 min at 37oC, but the present of ATP stabilizes it.Engelhardt (1941):EngelhardtATP proposed
13、to be the ATP proposed to be the molecule that promote molecule that promote muscle contraction (1942)muscle contraction (1942)lEngelhardt, W. A. (1942) Enzymatic and Mechanical Properties of Muscle Proteins Yale J Biol Med. 15: 2138. (translated)lOnly ATP was found to produce a marked effect on the
14、 myosin thread.ATP promotes muscle relaxation (not contraction).ATP promotes muscle relaxation (not contraction).Myosin: chemical transformer which converts chemical energy into mechanical action.Two forms of Myosins were revealed (1942)lMyosin A and myosin B from minced rabbit skeletal muscle. lMyo
15、sin B had a much higher relative viscosity which on addition of ATP was reduced to a value similar to that of Myosin AlThe viscosity of myosin A changed little by the addition of ATP.(in Hungary)Myosin B (renamed actomyosin) threads was observed to contract on addition of ATP! (1942)+ ATP (boiled mu
16、scle juice)+ ATP (boiled muscle juice)- ATP- ATPThread of Myosin BThe contribution of on muscle biology from the lab of Albert Szent-GyorgyilDiscovery is seeing what everybody else has seen, and thinking what nobody else has thought. Albert Szent-GyorgyilSzent-GyorgyiSzent-Gyorgyi(1893-1986)(1893-19
17、86) By Andrew Szent-GyorgyiNobel Prize in Physiology or Medicine, 1937:Nobel Prize in Physiology or Medicine, 1937:For his discoveries in connection with the biological For his discoveries in connection with the biological combustion processes, with special reference to combustion processes, with sp
18、ecial reference to vitamin C and to the catalysis of fumaric acid.vitamin C and to the catalysis of fumaric acid.( (both revelation was NOT correctboth revelation was NOT correct!) !)Actin was discovered as a second component in Myosin B (1942)lActin was identified as a second protein component in m
19、yosin B (Straub, 1942), the latter renamed as “actomyosin”.lIt transforms Myosin A into the contractile one, thus named as “actin”.lIt exists as globular or fibrous forms (G-actin and F-actin).The structure of myosin was examined via proteolytic cleavagelMihalyi E. & Szent-Gyorgyi AG. (1953) Trypsin
20、 digestion of muscle proteins. III. Adenosinetriphosphatase activity and actin binding capacity of the digested myosin. J Biol Chem. 201:211-9.lLowey et al. (1969) Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 42:1-29. lShort trypsin cleavage led
21、to decrease of viscosity and generate two fragments: “fast”(heavy) fraction containing the ATPase and actin binding activities.Sedimentation: the fast fraction binds actin!Digested myosin alone+ actin3 minutes afterreaching full speed.95 minutes afterreaching full speed.Isolated slow componentIsolat
22、ed fast componentForming fiber-likestructureATPase & actin bindingslowfastslowslowWith ATPaseNo ATPaseMyosin was revealed to contain two (not three) long polypeptide chainslSlayter HS, Lowey S. (1967) Substructure of the myosin molecule as visualized by electron microscopy. PNAS. 58:1611-8. lBiparti
23、te heads of high flexibility.l(single lobe after complete papain digestion)The Head structure of myosin was determined (1993)lRayment et al. (1993) Structure of the actin-myosin complex and its implications for muscle contraction. Science. 261:58-65.lRayment et al. (1993). Three-Dimensional Structur
24、e of Myosin Subfragment-1: A Molecular Motor. Science 261: 50 - 65.l RegulatoryDomain (lever arm)The myosin light chains are considered to stiffen the bound region of the heavy chainlOriginally thought to regulate the actin binding and ATPase activities.lMay function to stiffen the bound region of t
25、he heavy chain and thus to aid in force generation and transmission.Purified myosin and actin are able to form filaments under in vitro conditionsPurified myosin forms bipolar aggregates: with the tail stacking one on another.(Each is made of several hundred myosin molecules)Purified actin (G-actin)
26、 associates to form long filaments or F-actin. G-actinDetail EM structure analysis reveals regular thick-thin filament patterns (1953)lHANSON J, HUXLEY HE. (1953) Structural basis of the cross-striations in muscle. Nature. 172:530-2.lWhen myosin was extracted, the A-bands disappeared.lDuring stretch
27、, the length of A band does not change, but that of the I band shortens. before afterbefore afterHexogonal arrangement of thick and thin filamentsHugh E. HuxleyEM observations reveal the regular pattern change during muscle contraction and stretch (1954)lHuxley H & Hanson, J. (1954) Changes in the c
28、ross-striations of muscle during contraction and stretch and their structural interpretation. Nature. 173:973-6. lHuxley A & Niedergerke, R. (1954) Structural changes in muscle during contraction; interference microscopy of living muscle fibres. Nature. 173:971-3. lThe length of the I-band, but not
29、the A-band changes during muscle contraction and stretch (relaxation)! lActin filaments seem to slide out of or into the think filaments.The same fibril (of 4 sarcomeres)undergoing ATP-inducedcontraction under in vitro conditions.A-bandI-bandHugh E. Huxley Sir Andrew F Huxley(Nobel Prize 1963) A fil
30、ament sliding model was proposed to explain muscle contraction (1957)AnisotropicAnisotropicIsotropicIsotropicHuxley, A. F. (1957). Muscle structure and theories of contraction. Prog. Biophys.Biophys. Chem. 7: 255-318.Major observation: The A band maintained constant while the I band shortens during
31、the contraction process.Cross-bridges found to reorient during contraction-relaxation (1965)lReedy et al (1965) Induced Changes in Orientation of the Cross-Bridges of Glycerinated Insect Flight Muscle. Nature, 207: 1276 - 1279.Rigor relaxedRigor relaxed(-ATP +ATP)(-ATP +ATP)Rigor relaxedRigor relaxe
32、d(-ATP +ATP)(-ATP +ATP)Tropomyosin was isolated from muscle as an asymmetric protein (1946)lBailey, K. (1946) Tropomyosin: A new asymmetric protein component of muscle. Nature.157:368369.lBailey, K. (1948) Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem J. 43:271-9. Fib
33、rous: high viscosityFibrous: high viscosity(similar to myosin)(similar to myosin)homogeneoushomogeneousWith a size of about 90 kDa;With a size of about 90 kDa;X-ray diffraction pattern of the X-ray diffraction pattern of the a a a a-type;-type;Proposed to be the prototype of myosin (thus the name)Pr
34、oposed to be the prototype of myosin (thus the name)Crystals are formedCrystals are formedTroponin was revealed as a one which allows the natural actomyosin to be controlled by Ca2+ (1963) lEbashi, S. 1963. Third component participating in the superprecipitation of “natural actomyosin”.Nature. 200:1
35、010.lEbashi, S., and F. Ebashi. 1964. A new protein component participating in the superprecipitation of myosin B. J. Biochem. 55:604613.Superprecipitation of trypsin-treated “natural actomyosin” was Ca2+ insensitive;Became Ca sensitive with the newComponent added.New protein Component addedThe larg
36、est polypeptide, Titin was discovered in musclelWang et al (1979) Titin: Major myofibrillar components of striated muscle, PNAS, 76:3698.lPresent in M & Z lines, the junctions of A & I bands, and perhaps throughout the entire A bands.lWith about 27,000 amino acids.(c) SmoothmuscleImmunofluorescent s
37、taining of titin in chicken breast myofibrils (titin is present widely in sarcomere)Titin: Extends from the Z disk to the M disk. Titin is the largest polypeptide so far found in nature.Ig domainTroponin-C1) F-actin - a polymer of many G-actin monomersF-ActinTropomyosinTroponin-ITroponin-TG-Actin mo
38、nomer2) Tropomyosin （原肌球蛋白）（原肌球蛋白） - lies in the groove between F-actin strands and blocks myosin binding sites on actin.3) Troponins (肌钙蛋白；肌钙蛋白；Tn): Tn-I binds to actin & inhibits actomyosin interaction: Tn-T binds to tropomyosin;Tn-C binds to Ca2+. A complex of multiple interacting proteins on F-a
39、ctin regulate myosin-actin interaction.Other proteins found in musclelThey form the Z disk (e.g., a a-actinin, desmin, and vimentin) and the M disk (e.g., paramyosin, C-protein, and M-protein); organize the arrays of thick and thin filaments (e.g., titin and nebulin); regulate myosin-actin interacti
40、on (e.g., tropomyosin and troponin).lNebulin (伴肌动蛋白伴肌动蛋白), having 7,000 residues, is thought to be structures as an a helix spending the length of the thin filament.The current model of muscle contractionlMyosin with bound ATP has a low affinity for actin (ATP dissociates the actomyosin interaction)
41、, but the “primed” head (with bound ADP and Pi) binds readily.lThe release of Pi from myosin provokes a major rotation, or “power stroke,” of the lever arm. lDissociation of ADP from myosin generate a change of conformation for the myosin head. lThe mechanism of transduction of chemical energy, deri
42、ved from ATP hydrolysis, into mechanical work was detailed in the “power stoke” model (1971).Calcium attaches to troponin/ tropomyosin; they roll away, exposing the active site on actin.Myosin cross-bridges attach to active site on actin.After attachment, the cross-bridges pivot, pulling the thin fi
43、laments.A fresh ATP replaces the ADP+Pi, allowing myosin and actin to detach.Energy from the splitting of the fresh ATP allows repositioning of the myosin head.This leads back to Step 1, which continues the cycle as long as calcium ions are attached to troponin/tropomyosin.Contraction of skeletal mu
44、scleThe muscle fiber is stimulated.Ca2+ ions are released.Thin filaments move to middle of sarcomere.Muscle fiber contracts.Muscle tension increases.The molecular understanding of contraction of the skeletal muscle provides a paradigm for understanding the molecular process of other motions in the living organisms
