Todays Lecture

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Todays Lecture

• 15) Friday, November 3 Applications
• Restrained Molecular Dynamics
• Ligand binding

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Putting it all together

• We have learned a lot of things in a short period
  of time
• Behavior of nuclear spins in a magnet (Bloch
  equations)
• Sampling of NMR data and Fourier transform
• Rules of interactions of 2 spins (product
  operators)
• 2D NMR experiments
• J-coupling (Karplus equation and dihedral angles)
• NOE (molecular ruler)
• Relaxation and dynamics

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What do you do with all of it???

• With NMR you can learn about
• Molecular structure
• Molecular dynamics
• Ligand-receptor interactions
• Interacting proteins
• Protein folding
• Enzyme catalysis

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NMR structures
NMR structure determination is quite straight
forward. The mainstay of the work is the NOE,
but J-coupling can play a big role as well.
Residual dipolar couplings can significantly
improve the quality of NMR structures. The first
step in a peptide or protein NMR analysis is
sequential resonance assignments. This was
covered in the lecture on TOCSY and NOESY for 1H
assignments and in the lecture on HSQC and triple
resonance experiments for 1H, 15N, and 13C
protein assignments.
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NMR structures
Following assignments, pairwise NOE interactions
are measured.
Protein primary amino acid sequence
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NMR structures
Following assignments, pairwise NOE interactions
are measured. NOEs are sometimes all that are
used for NMR structures, but J-couplings can help
define local geometries around dihedral angles.
Dipolar couplings orient bonds in space.
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20 amino acid Mini Protein
Some regions are well-defined
This is a family of 38 structures from a
simulated annealing and energy minimization
calculation.
Other regions are disordered
Neidigh, Fesinmeyer, and Andersen Designing a
20-residue protein Nature Structural Biology 9,
pp 425-430 (2002).
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Why a family of structures?

• NMR structures are solved using variants of 2
  main methods
• Restrained molecular dynamics (RMD) or simulated
  annealing (SA)
• RMD uses NMR restraints (NOEs, J-couplings,
  dipolar couplings) as extra pseudopotentials in
  a molecular dynamics simulation (Fma). A family
  of structures can be made from time points along
  the MD trajectory. Simulated annealing uses RMD
  but changes the temperature of the simulation
  from hot to cold in order to try to get to a
  global minimum.
• Distance geometry (DG) is a geometrical method
  that simultaneously finds the solution to all the
  measured pairwise distances. This is repeated
  multiple times, and the structures from these
  different calculations are superimposed as a
  family.
• Each member in a family of structures satisfies
  the NMR data. By showing several structures,
  regions that are well-defined can be
  distinguished from those that are poorly defined.

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What do the fuzzy regions mean?
Either flexibility, not enough data, or several
fixed conformations
This is difficult or impossible to answer without
relaxation measurements. They might be real
motion but they might also be well-structured
regions that stick out and have no NOEs to the
rest of the protein. Dipolar couplings can help
sort these out.
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Ligand binding by NMR depends on the exchange rate

• Fast or intermediate exchange Saturation
  transfer
• Slow exchange Isotope edited experiments

A. S. Edison University of Florida
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Ligand binding by NMR depends on the exchange rate
kex1 s-1
If two NMR peaks are in chemical exchange, the
spectrum depends significantly on the exchange
rate. The simulations on the left are two peaks
separated by 80 Hz and with different exchange
rates. Note that when the exchange rate is close
to the difference in frequencies, the peaks
almost disappear.
kex80 s-1
kex180 s-1
kex250 s-1
kex1000 s-1
kex5000 s-1
A. S. Edison University of Florida
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SAR by NMR
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SAR by NMR
N (ppm)
15
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Line broadening methods
2-phenoxybenzoic acid binding to P38 MAP kinase.
Nicotinic acid does not bind
Lepre et al., 2004
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Saturation Transfer
Lepre et al., 2004
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Saturation Transfer
Lepre et al., 2004
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Saturation Transfer Difference
Lepre et al., 2004
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Saturation Transfer
Lepre et al., 2004
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The End
All outstanding homework must be turned in next
Wed, Nov 8 (LG-187). Reports for the class and
lab due no later than Monday, Nov 13 (LG-187)
A. S. Edison University of Florida