Protein Dynamics from NMR

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Protein Dynamics from NMR
03/19/02
Amide proton exchange Heteronuclear
relaxation Application to determine the mechanism
of cooperativity in binding of Ca2 by calbindin
D9k
Protein and Peptide Drug Analysis, pages 714-716
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Why The Interest In Dynamics?

• Function requires motion/kinetic energy
• Entropic contributions to binding events
• Protein Folding/Unfolding
• Uncertainty in NMR and crystal structures
• Effect on NMR experiments- spin relaxation is
  dependent on rate of motions ? know dynamics to
  predict outcomes and design new experiments
• Quantum mechanics/prediction (masochism)

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Characterizing Protein Dynamics
Parameters/Timescales
Relaxation
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NMR Parameters That Report On Dynamics of
Molecules

• Number of signals per atom multiple signals for
  slow exchange between conformational states
• Linewidths narrow faster motion, wide
  slower dependent on MW and conformational states
• Exchange of NH with solvent requires local
  and/or global unfolding events ? slow timescales
• Heteronuclear relaxation measurements
• R1 (1/T1) spin-lattice- reports on fast motions
• R2 (1/T2) spin-spin- reports on fast slow
• Heteronuclear NOE- reports on fast some slow

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Relaxation- Return to Equilibrium
t
t
z axis
x,y plane
0
0
Longitudinal
Transverse
1
1
t
t
2
2
E-t/T2
1-e-t/T1
8
8
Transverse always faster!
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Longitudinal (T1) Relaxation

• MECHANISM
• Molecular motions cause the nuclear magnets to
  fluctuate relative to a fixed point in space
• Fluctuating magnetic fields promote spins to flip
  between states Induced by the lattice!!
• Over time, spin flips cause a return to
  equilibrium
• Slow motions make effect more efficient

Slow
Fast
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Transverse (T2) Relaxation

• MECHANISM
• Magnetic field is not homogenous to an infinite
  degree
• Each spin comprising the bulk magnetization will
  feel a slightly different field
• Over time, the spin fan out (lose coherence)
• Slow motions make effect more efficient

t
Slow
Fast
dMx,y/dt Mx,y/T2 ?Linewidth
time
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Linewidth is Dependent on MW

• Linewidth determined by size of particle
• Fragments have narrower linewidths

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Amide Proton Exchange(sec?min?hours?days?months)

• Peptides/unfolded proteins exchange rapidly
• Folded proteins protected solvent accesibility,
  H-bonds

• H-bonded amides exchange occurs via local or
  global unfolding events

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Heteronuclear Relaxation(psec?nsec msec?msec)

• 15N relaxation dominated by 1H
• N-H distance fixed, variation in relaxation due
  to differences in motional properties
• Overall tumbling, internal motions

• Must fit relaxation parameters to a motional
  model Lipari-Szabo order parameter (S2) most
  common

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Dynamics To Probe The OriginOf Structural
Uncertainty
?

• Measurements show if high RMSD is due to high
  flexibility (low S2)

Weak correlation
?
?
Strong correlation
?
?