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The enzymology of chemo-mechanical energy transduction Motors; “*-dependent” NTPases

The enzymology of chemo-mechanical energy transduction Motors; “*-dependent” NTPases. Biophysical Society Summer Course 11 July 2012 Charlie Carter. Readings. Nelson, P., Biological Physics, Chapter 10

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The enzymology of chemo-mechanical energy transduction Motors; “*-dependent” NTPases

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  1. The enzymology of chemo-mechanical energy transductionMotors; “*-dependent” NTPases Biophysical Society Summer Course 11 July 2012 Charlie Carter

  2. Readings • Nelson, P., Biological Physics, Chapter 10 • Howard, Jonathan, Mechanics of Motor Proteins and the Cytoskeleton, Sinauer Associates, Sunderland, MA • Chapter 12 Structures of Motor Proteins • Chapter 14 ATP Hydrolysis • Chapter 16 Motility Models

  3. Questions • What does “Transduce” mean? • Why is NTP hydrolysis so special? • It is quite slow in water; needs a catalyst! • It explosively exergonic (ie., favorable) in water! • How does water change the equilibrium constant for NTP hydrolysis? • Why are pre-steady state and steady state rates different? • What does “energy storage” mean? • What does it mean when product release is rate limiting? • Examples of coupling: • Myosin cross-bridge cycle: an actin-dependent ATPase • F1 ATPase cycle: a work-dependent ATP synthase. • Kinesin cycle: a tubulin-dependent ATPase • GroElEs cycle: an improperly folded protein-dependent ATPase • RAS cycle: a signaling GTPase with two dependencies

  4. Transduction (from the OED) transduce (tr":ns£dju:s, trÊns-, -nz-), v. 1. trans.To alter the physical nature or medium of (a signal); to convert variations in (a medium) into corresponding variations in another medium.

  5. ATP + H2O ADP + Pi Keq >> 1.0 … Becomes reversible inside a protein that can absorb the explosion... Keq ~ 1.0 ATP + H2O ADP + Pi NTP hydrolysis fuels everything in the cell! A reaction that is explosivelyirreversible in water… …by changing shape, which stores free energy. These shape-changes drive all cellular processes!

  6. DG = 0 For a complete cycle Binding Equilibria A thermodynamic cycle with an labile substrate => 3 states! Conformational Equilibria

  7. Motors F1 ATPase Hydrolysis Chemical transformation of nucleotide Synthesis Chemical transformation of nucleotide Work in Product release NTP binding Nucleotide exchange Turnover Induced fit NDP binding Nucleotide exchange Work out Product (ADP, Pi) release 3-State behavior and free energy transduction Closed, Triphosphate Keq ~ 1 !!! Induced fit Catalysis Turnover Open, Ligand-free Closed, diphosphate

  8. Free solution Tubulin subunit Microtubule Tubulin thermodynamic cycles show Keq ~0 NTP NDP + Pi Caplow, Ruhlen, & Shanks (1995) J. Cell Biol., 127:779-788

  9. Perchloric acid quench The quench-flow technique: perchloric acid Enz S

  10. 20/s Myosin vs Actomyosin 0.1/s Steady-state ~100/s Transient phase Ed Taylor: energy transduction revealed

  11. Howard, J. (2001) Mechanics of Motor Proteins and the Cytoskeleton, Ch. 14

  12. X-ray kinetics correlates cross-bridge activity, tension

  13. Work is done only when cross-bridges are attached Length of power stroke

  14. The amount of work done each cycle depends on how much is lost in vertical drops! T. Hill’s account of the actomyosin free energy cycle

  15. Ron Milligan’s myosin movie

  16. Ron Milligan’s kinesin movie

  17. Differences between myosin, kinesin ATPases J. Howard, Mechanics of Motor Proteins and the Cytoskeleton

  18. Stator (unknown) Rotor (F0) ATP Synthase • CS3 and CS38 • Solved in pieces: F1,F0 • Nobel Prize (Chemistry) 1997

  19. Translocating protons down a gradient can drive rotaty motion: molecular motors

  20. aTP-subunit Non-exchangeable ATP bE-subunit g-subunit, N-terminal helix

  21. Strand 3 B-helix

  22. Why don’t the examiners pose questions to candidates other than in a twisted manner? It seems that they fear being understood by those they are interrogating; what is the origin of this deplorable habit of complicating the questions with artifical difficulties? -Evariste Galois, French Mathematician, inventor of Group Theory

  23. Study Questions • Use the data on slide #12 to calculate the Keq for ATP hydrolysis within the Myosin Active site. • Use slide #21 to discuss why there has to be an elastic component for any working motor to be at all efficient. • AMPPNP is often thought to be a “non-hydrolyzable” ATP analog. Yet, it drives the accumulation of Ca2+ by the sarcoplasmic reticulum pump. Use these ideas to deconstruct the next sentence. In skeletal muscle fibers depleted of ATP (Rigor), AMPPNP causes a: • Rapid, fully reversible, stress-independent increase in the rest length • Whilst the Isotonic stiffness remains within 2% of the Rigor value. • Use your answer to the previous question to discuss how, if primates had prehensile tails consisting largely of thin and thick filaments might be able to synthesize ATP by bungi jumping.

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