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

Copyright, C. W. Carter, Jr

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

Biophysical Society Summer Course11 July 2012

Charlie Carter


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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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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


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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.


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ATP + H2O ADP + Pi

A reaction that is explosively irreversible in water…

… Becomes reversible inside a protein that can absorb the explosion...

ATP + H2O ADP + Pi

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


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A thermodynamic cycle with an labile substrate => 3 states!

ConformationalEquilibria

BindingEquilibria

G = 0For a complete cycle+


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NTP bindingNucleotide exchange

HydrolysisChemical transformation of

nucleotide

Work outProduct (ADP, Pi) release

Motors

Work inProduct release

SynthesisChemical transformation of

nucleotide

NDP bindingNucleotide exchange

Turn

over

Induced fit

F1 ATPase

Open,Ligand-free

Closed,Triphosphate

3-State behavior and free energy transduction

Closed,diphosphate

Indu

ced

fit Catalysis

Turnover

Keq ~ 1 !!!


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NTP

NDP + Pi

Tubulin thermodynamic cycles show Keq ~0

Free solution Tubulin subunit Microtubule

Caplow, Ruhlen, & Shanks (1995) J. Cell Biol., 127:779-788


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The quench-flow technique: perchloric acid

Perchloric acid quench

Enz

S


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Ed Taylor: energy transduction revealed

Steady-state0.1/s

20/s

Transient phase~100/s

Myosin vs Actomyosin


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


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X-ray kinetics correlates cross-bridge activity, tension


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Work is done only when cross-bridges are attached

155 Å

155 Å

110 Å

110 Å = 155 sin 45

Length of power stroke


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T. Hill’s account of the actomyosin free energy cycle

-110 Å 0 Å X

45

90 o

o

Rigor ==> min G |

potential

potential

M**D + T

M*D + T

AM*D + T

M*D + DAM* + D

AM**D + T

10

0

G(kcal/M)

The amount of work done each cycle depends on how much is lost in vertical drops!


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Ron Milligan’s myosin movie


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Ron Milligan’s kinesin movie


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Differences between myosin, kinesin ATPases

Myosin Kinesin

Product (ADP) ReleaseStrengthens actin

bindingExhange Promotes

power stroke

Release from trackPromoted by ATP

bindingPromoted by ATP

hydrolysis

ATP hydrolysisWhile Detached

from actinWhile Bound to T

Rate limiting stepOccurs while

detachedOccurs while

attached

Duty Ratio (%time attached) 0.035 - 0.14 0.5 - 1.0

Cross-bridge stiffness 5pN/nm ~0.5pN/nm

Speed 6000 nm/s 800 nm/s

J. Howard, Mechanics of Motor Proteins and the Cytoskeleton


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ATP Synthase

• CS3 and CS38• Solved in pieces: F1,F0• Nobel Prize (Chemistry) 1997

Stator(unknown)

Rotor (F0)


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Translocating protons down a gradient can drive rotaty motion: molecular

motors


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-subunit,N-terminal helix

-subunitTP-subunit

Non-exchangeable ATP


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B-helixStrand 3


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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


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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.

Documents

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