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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 - PowerPoint PPT Presentation
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Copyright, C. W. Carter, Jr
UNCrystallographers
The enzymology of chemo-mechanical energy transductionMotors; “*-dependent” NTPases
Biophysical Society Summer Course11 July 2012
Charlie Carter
Copyright, C. W. Carter, Jr
UNCrystallographers
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
Copyright, C. W. Carter, Jr
UNCrystallographers
Copyright, C. W. Carter, Jr
UNCrystallographers
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
Copyright, C. W. Carter, Jr
UNCrystallographers
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.
Copyright, C. W. Carter, Jr
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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
Copyright, C. W. Carter, Jr
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A thermodynamic cycle with an labile substrate => 3 states!
ConformationalEquilibria
BindingEquilibria
G = 0For a complete cycle+
Copyright, C. W. Carter, Jr
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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 !!!
Copyright, C. W. Carter, Jr
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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
Copyright, C. W. Carter, Jr
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Howard, J. (2001) Mechanics of Motor Proteins and the Cytoskeleton, Ch. 14
Copyright, C. W. Carter, Jr
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Copyright, C. W. Carter, Jr
UNCrystallographers
Copyright, C. W. Carter, Jr
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X-ray kinetics correlates cross-bridge activity, tension
Copyright, C. W. Carter, Jr
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Copyright, C. W. Carter, Jr
UNCrystallographers
Copyright, C. W. Carter, Jr
UNCrystallographers
Copyright, C. W. Carter, Jr
UNCrystallographers
Copyright, C. W. Carter, Jr
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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!
Copyright, C. W. Carter, Jr
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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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Copyright, C. W. Carter, Jr
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-subunit,N-terminal helix
-subunitTP-subunit
Non-exchangeable ATP
Copyright, C. W. Carter, Jr
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B-helixStrand 3
Copyright, C. W. Carter, Jr
UNCrystallographers
Copyright, C. W. Carter, Jr
UNCrystallographers
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
Copyright, C. W. Carter, Jr
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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.