From structure to function

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.

very slow slow fast very fast ultrafast

SLOW s ms s ns ps fs FAST

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?

Chemical reaction, conformational equilibrium, exchange between the bound and

free states of a ligand/protein complex, etc.

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

Two basic approaches

–  Monitor changes in target protein due to binding of ‘ligand’ (small molecule,
protein, nucleic acid, lipid, sugar, etc.)
–  Detect specific interaction ‘epitopes’ on ligand

target ligand
protein differences in chemical shifts, H/D
exchange rates, NOE intensities

•  Parameters sensitive to changes in binding site occupancy

–  Chemical shift
–  Amide H/D exchange rates
–  NOE spin diffusion across interface
•  NMR experiments used:
–  HSQC (shift perturbation or H/D exchange)
–  NOE difference (either 1D or 2D)
 Slow exchange vs fast exchange

Normally binding is either fast or slow exchange or in between.

At intermediate conditions, different peaks in the same spectrum can display both
types, depending on shift differences
For binding reactions, slow exchange
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Before we attempt to study the structure of a ligand in a complex, it
is useful to determine if the complex is in fast or slow exchange on
the NMR timescale. This is often done by titrating the ligand (L) into
a sample containing the receptor (R) and monitoring the NMR
spectrum:
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-0&1(+2,3%'4+ .%-#(+2,3%'4+
567/587

produces distinct signals for free and 9/:

bound states at intermediate titration

9;</:
points - follow binding reaction by
watching bound/free peak intensities :/:
grow/diminish
:;</:

Fast exchange - only one set of peaks =/:

throughout titration, shifting in proportion to 8. 8> 6. 6> 8. 8> 6. 6>

changing ratio of free:bound
As a rough approximation:
• tight binding (slow exchange) is characterized by KD < M
• weak binding (fast exchange) is characterized by KD > M
Is this binding slow or fast on the NMR scale?
What information can we extract from the titration data?

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

Comparison of the binding sites of the FB–Fc complex

B domain of staphylococcal protein A (FB) complexed with the Fc fragment of immunoglobulin G (IgG)

X-ray Chemical shift H/D exchange

 Saturation Transfer Difference NMR (STD NMR)
STD NMR experiments detect magnetization that is transferred from a receptor
protein to a bound ligand. Only bound ligands show STD effects.

STD NMR is extremely robust and gives maximal effects at protein to ligand ratios
greater than ca. 1:100.

Less than 1 nM of protein is necessary for screening.

The dissociation constant should be in the range between nM and mM.

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.