Transcript Center for Structural Biology

03/19/02
Protein Dynamics from NMR
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
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)
Characterizing Protein Dynamics:
Parameters/Timescales
Relaxation
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
Relaxation- Return to Equilibrium
t
t
x,y plane
Transverse
0
Longitudinal
1
1
t
t
2
2
8
E-t/T2
1-e-t/T1
Transverse always faster!
8
0
z axis
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
dMz/dt = Meq – Mz/T1
t
Mz(t) = Meq (1-e-t/T1)
Slow
Fast
Mz(t)  Meq
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
time
Slow
Fast
dMx,y/dt = Mx,y/T2
Linewidth
Linewidth is Dependent on MW
A
B
A
B
Big
Small
(Slow) (Fast)
15N
Linewidth
determined by
size of particle
15N
15N
Fragments
have narrower
linewidths
1H
1H
1H
Amide Proton Exchange
(secminhoursdaysmonths)
-NH
OH-N
•Peptides/unfolded
proteins exchange
rapidly
•Folded proteins
protected: solvent
accesibility, H-bonds
H-bonded amides: exchange occurs via local or
global unfolding events
Heteronuclear Relaxation
(psecnsec & msecmsec)
•15N relaxation
dominated by 1H
-15NH
•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
Dynamics To Probe The Origin
Of Structural Uncertainty

Weak correlation
Strong correlation 


Measurements
show if high RMSD
is due to high
flexibility (low S2)