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Protein Dynamics from NMR

03/19/02. Protein Dynamics from NMR. Amide proton exchange Heteronuclear relaxation Application to determine the mechanism of cooperativity in binding of Ca 2+ by calbindin D 9k. Protein and Peptide Drug Analysis, pages 714-716. Why The Interest In Dynamics? .

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Protein Dynamics from NMR

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  1. 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

  2. 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)

  3. Characterizing Protein Dynamics: Parameters/Timescales Relaxation

  4. 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

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

  6. dMz/dt = Meq – Mz/T1 Mz(t) = Meq (1-e-t/T1) t Mz(t)  Meq 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

  7. 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

  8. B A B A Big (Slow) Small (Fast) 15N 15N 15N 1H 1H 1H Linewidth is Dependent on MW • Linewidth determined by size of particle • Fragments have narrower linewidths

  9. D-O-D -N- H OH-N 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

  10. -15N- -15N- -15N- H H H 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

  11. Dynamics To Probe The OriginOf Structural Uncertainty  Weak correlation • Measurements show if high RMSD is due to high flexibility (low S2)   Strong correlation  

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