NMR in Drug Design
NMR in Drug Design
NMR in Drug Design
Lecture 6
Bimolecular NMR in:
Proteins
Nucleic Acids
Protein-Nucleic Acids
Protein-membrane/lipid
Polysaccharides
Protein-Protein interactions
Advantages:
1) Assess direct binding
2) Identify the binding regions
3) Determine the binding affinity
4) Assess protein folding upon binding to another partner
Limitations:
1) Size of the complex
2) Relaxation and tumbling (leads to signal broadening)
How much Information you can get from an NMR experiment of protein-protein
complex?
direct binding
binding interface
affinity
Protein Dynamics
So far we have talked about techniques and experiments used to study ‘one form’ of molecules by NMR.
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Dynamic NMR (DNMR) deals with the effects “in a broad sense” of chemical exchange processes on NMR
spectra.
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NMR is unique in the range of such processes that it can usefully provide detailed mechanistic and kinetic
information about reactions that are occurring in equilibrium mixtures.
MACROSCOPIC
DIFFUSION,
FLOW
CHEMICAL
EXCHANGE
MOLECULAR
ROTATIONS
MOLECULAR
VIBRATIONS
3 2 1
RELAXATION SPECTRAL LARMOR
TIMESCALE TIMESCALE TIMESCALE
Study of dynamic processes by NMR
What if a molecule is undergoing some dynamic process?
Examples?
Conformational Kex
equilibrium
Ka
Chemical
equilibrium Kd
Correlation:
Affinity
NMR scale
Tight binding
Fast Exchange
Moderate
Transient exchange
Weak binding
Slow exchange
Chemical exchange vs. NMR timescale
T (K)
273
263
253
243
223
• At coalecense temperature: the rate of the exchange between the two species
becomes comparable to the difference in chemical shifts of the sites.
• NMR measures rates from 10-6 to 108 s-1.
Binding studies by NMR
target ligand
protein differences in chemical shifts, H/D
exchange rates, NOE intensities
o)2 + (Y-Yo)2
Δδ = √(X-X
Slow exchange
Protein (black)
0.5:1 ligand:protein (magenta)
1:1 ligand:protein (orange).
Amide exchange rates
t = 0 - No D2O
15N
Add D2O
15N
t = t1
t = t2
15N
8.0
(NHs)
7.0
H/D exchange interface
mapping
• Protein A binding to B
• Measure D2O exchange rates
on free Protein A
• Form complex in H2O, dilute
into D2O, exchange for varying
times, dissociate complex with
low pH
• Collect spectra on free Protein
A after exchange period.
NMR Study of the Interaction between the B Domain of Staphylococcal Protein A and the Fc Portion
of Immunoglobulin G Gouda, H.; Shiraishi, M.; Takahashi, H.; Kato, K.; Torigoe, H.; Arata, Y.; Shimada, I.;
Biochemistry 1998; 37(1); 129-136.
• Cross-saturation gives best correlation to binding interface defined in x-ray structure
• Chemical shift and H/D exchange methods both suffer from indirect effects of
conformational/dynamic changes
B domain of staphylococcal protein A (FB) complexed with the Fc fragment of immunoglobulin G (IgG)
STD NMR is extremely robust and gives maximal effects at protein to ligand ratios
greater than ca. 1:100.
free L
Ligand-bound
protein
protein
Saturation of the protein leads to a direct saturation of those parts of ligand(s) in
direct contact to the protein. By exchange between bound and free state the
saturation is transported to solution and detected by subtracting a spectrum with
saturation from a normal spectrum.
STD NMR gives precise information about the binding epitope of the ligand. This is
very important information for the design of a potent drug. The optimal drug is of
optimal size and optimal shape.