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1、Myosin 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 division; organelle streaming, etc.Biochemistry Lecture for Oct 16, 2012EMstudiesB
2、iochemical studieslMany things are moving in living organisms, e.g.:Muscle contraction in vertebrates (myosin and actin);Migration of organelles along microtubules and chromosome separation in dividing cells (kinesins and dyneins);Rotation of bacterial flagella (a complex rotational motor proteins);
3、DNA metabolism (helicases and polymerases).lVia cyclic conformational changes of proteins, consuming chemical energy supplied by ATP.lAchieving exceptionally high levels of spatial and temporal organization.Striated appearance: Alternating light and dark bands;I-band: Zone of thin filaments (of acti
4、n) alone; Z-line: the middle of thin filaments;A-band: Entire length 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!) Anisotro
5、picisotropicLeeuwenhoek first discovered such muscle cross-striations in 1682! proteins fromskeletal muscle; & smooth muscle (c)Electron micrograph of a longitudinal section of skeletal muscle fiber.Cross sectionsCycle stops here in relaxed living muscle (due to removal of myoplasmic calcium)Ca2+*Hi
6、gh actin affinityADP+PiADP+PiPiADP*Low actin affinity*ATPATPResting musclePower strokelA viscous protein 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 “en
7、zyme”).lMuralt & Edsall (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
8、double refraction.lEngelhardt 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):EngelhardtlEngelhardt, W. A. (1942
9、) 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 myosin thread.Myosin: chemical transformer which converts chemical energy into mechanical action.lMyosin A and myosin B from minced rabbit skeletal mu
10、scle. lMyosin 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)Thread of Myosin BlDiscovery is seeing what everybody else has seen, and thinking what nobody e
11、lse has thought. Albert Szent-Gyorgyil By Andrew Szent-GyorgyilActin was identified as a second protein component in myosin 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 an
12、d F-actin).lMihalyi E. & Szent-Gyorgyi AG. (1953) Trypsin 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 degra
13、dation. J Mol Biol. 42:1-29. lShort trypsin cleavage led 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 minut
14、es afterreaching full speed.Isolated slow componentIsolated fast componentForming fiber-likestructureATPase & actin bindingslowfastslowslowWith ATPaseNo ATPaselSlayter HS, Lowey S. (1967) Substructure of the myosin molecule as visualized by electron microscopy. PNAS. 58:1611-8. lBipartite heads of h
15、igh flexibility.l(single lobe after complete papain digestion)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 Structure of Myosin Subfragment-1: A Molecular Motor. Science 261: 50 -
16、 65.l RegulatoryDomain (lever arm)lOriginally thought to regulate the actin binding and ATPase activities.lMay function to stiffen the bound region of the heavy chain and thus to aid in force generation and transmission.Purified myosin forms bipolar aggregates: with the tail stacking one on another.
17、(Each is made of several hundred myosin molecules)Purified actin (G-actin) associates to form long filaments or F-actin. G-actinlHANSON 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, the
18、length of A band does not change, but that of the I band shortens. before afterbefore afterHexogonal arrangement of thick and thin filamentsHugh E. HuxleylHuxley H & Hanson, J. (1954) Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation. Nature
19、. 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 the A-band changes during muscle contraction and stretch (relaxation)! lActin filaments seem to slid
20、e 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) Huxley, A. F. (1957). Muscle structure and theories of contraction. Prog. Biophys.Biophys. Chem. 7: 255-3
21、18.Major observation: The A band maintained constant while the I band shortens during the contraction process.lReedy et al (1965) Induced Changes in Orientation of the Cross-Bridges of Glycerinated Insect Flight Muscle. Nature, 207: 1276 - 1279.lBailey, K. (1946) Tropomyosin: A new asymmetric protei
22、n component of muscle. Nature.157:368369.lBailey, K. (1948) Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem J. 43:271-9. lEbashi, S. 1963. Third component participating in the superprecipitation of “natural actomyosin”.Nature. 200:1010.lEbashi, S., and F. Ebashi. 1964.
23、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 addedlWang et al (1979) Titin: Major myofibrillar c
24、omponents 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 staining of titin in chicken breast myofibrils (titin is present widely in sarcomere)Titin: Ex
25、tends 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 monomer2) Tropomyosin （原肌球蛋白）（原肌球蛋白） - lies in the groove between F-actin strands and blocks my
26、osin binding sites on actin.3) Troponins (肌鈣蛋白；肌鈣蛋白；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-actin regulate myosin-actin interaction.lThey form the Z disk (e.g., a a-actinin, desmin, and
27、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 interaction (e.g., tropomyosin and troponin).lNebulin (伴肌動蛋白伴肌動蛋白), having 7,000 residues, is thought to be structures as an a helix
28、 spending the length of the thin filament.lMyosin with bound ATP has a low affinity for actin (ATP dissociates the actomyosin interaction), 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. lDiss
29、ociation of ADP from myosin generate a change of conformation for the myosin head. lThe mechanism of transduction of chemical energy, derived from ATP hydrolysis, into mechanical work was detailed in the “power stoke” model (1971).Calcium attaches to troponin/ tropomyosin; they roll away, exposing t
30、he active site on actin.Myosin cross-bridges attach to active site on actin.After attachment, the cross-bridges pivot, pulling the thin filaments.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 muscleThe muscle fiber is stimulated.Ca2+ ions are released.Thin filaments move to middle of sarcomere.Muscle fiber contracts.Muscle tension increases.
生物化學(xué)：Chapter 5-4 Protein Function
