1. Copyright, C. W. Carter, Jr The enzymology of chemo-mechanical energy transduction Motors; “*-dependent” NTPases Biophysical Society Summer Course 11 July 2012 Charlie Carter

2. Copyright, C. W. Carter, Jr 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

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4. 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

5. Copyright, C. W. Carter, Jr 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.

6. Copyright, C. W. Carter, Jr ATP + H 2 O ADP + P i A reaction that is explosively irreversible in water… … Becomes reversible inside a protein that can absorb the explosion... ATP + H 2 O ADP + P i NTP hydrolysis fuels everything in the cell! …by changing shape, which stores free energy. These shape-changes drive all cellular processes! Keq ~ 1.0 Keq >> 1.0

7. Copyright, C. W. Carter, Jr A thermodynamic cycle with an labile substrate => 3 states! Conformational Equilibria Binding Equilibria  G = 0 For a complete cycle

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

9. Copyright, C. W. Carter, Jr NTP NDP + Pi Tubulin thermodynamic cycles show Keq ~0 Free solutionTubulin subunitMicrotubule Caplow, Ruhlen, & Shanks (1995) J. Cell Biol., 127:779-788

10. Copyright, C. W. Carter, Jr The quench-flow technique: perchloric acid Perchloric acid quench EnzEnz S

11. Copyright, C. W. Carter, Jr Ed Taylor: energy transduction revealed Steady-state 0.1/s 20/s Transient phase ~100/s Myosin vs Actomyosin

12. Copyright, C. W. Carter, Jr Howard, J. (2001) Mechanics of Motor Proteins and the Cytoskeleton, Ch. 14

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

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

21. Copyright, C. W. Carter, Jr T. Hill’s account of the actomyosin free energy cycle The amount of work done each cycle depends on how much is lost in vertical drops!

22. Copyright, C. W. Carter, Jr Ron Milligan’s myosin movie

23. Copyright, C. W. Carter, Jr Ron Milligan’s kinesin movie

24. Copyright, C. W. Carter, Jr Differences between myosin, kinesin ATPases MyosinKinesin Product (ADP) Release Strengthens actin binding Exhange Promotes power stroke Release from track Promoted by ATP binding Promoted by ATP hydrolysis ATP hydrolysis While Detached from actin While Bound to  T Rate limiting step Occurs while detached Occurs while attached Duty Ratio (%time attached) 0.035 - 0.140.5 - 1.0 Cross-bridge stiffness 5pN/nm~0.5pN/nm Speed6000 nm/s800 nm/s J. Howard, Mechanics of Motor Proteins and the Cytoskeleton

25. Copyright, C. W. Carter, Jr ATP Synthase CS3 and CS38 Solved in pieces: F1,F0 Nobel Prize (Chemistry) 1997 Stator (unknown) Rotor (F 0 )

26. Copyright, C. W. Carter, Jr Translocating protons down a gradient can drive rotaty motion: molecular motors

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28.  -subunit, N-terminal helix   -subunit  TP -subunit Non-exchangeable ATP

29. Copyright, C. W. Carter, Jr B-helix Strand 3

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31. 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

32. Copyright, C. W. Carter, Jr Study Questions l Use the data on slide #12 to calculate the Keq for ATP hydrolysis within the Myosin Active site. l Use slide #21 to discuss why there has to be an elastic component for any working motor to be at all efficient. l AMPPNP is often thought to be a “non-hydrolyzable” ATP analog. Yet, it drives the accumulation of Ca 2+ 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. l 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.