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Using NMR spectroscopy to elucidate the structure,
dynamics and interactions of proteins
Malene Ringkjøbing Jensen
Institut de Biologie Structurale
Grenoble, France
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One-dimensional 1H NMR spectrum of a protein (100 amino acids)
1H (ppm)
Methyl protons
H2O
Side chainprotons
Haprotons
Aromaticprotons
Backboneamide protons
Indole NH of Trp
• Typical experimental conditions: 100 µM - 1mM of protein in 150uL sample volume
• 1D 1H spectra of proteins offer only limited resolution
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3D NMR experiments based on transfer through scalar couplings
• The HNCO experiment correlates the
chemical shifts of 1H, 15N and 13C’ (of the
previous amino acid)
• Experiment is again based on transfer of
magnetization across the scalar couplings
(1H-15N and 15N-13C’)
• Typical experimental times:
1D 1H – minutes
2D 1H-15N – hours
3D 1H-15N-13C - days
Labeling of protein required: 13C, 15N
A set of 3D experiments for protein backbone assignment
Assignment procedure: correlating 13C’, 13Ca and 13Cb frequencies
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Ser Asp His ValAsp His
ω2 -13
C' (
ppm
)ω
2 -13
Cɑ (
ppm
)
ω2 -13
Cβ (
ppm
)
ω3 -1HN (ppm) ω3 -1HN (ppm)
i i - 1 i i - 1 i i - 1Ser Asp His ValAsp Hisi i - 1 i i - 1 i i - 1
A
B
C
The amino acids are linked one by one by simultaneously matching the three 13C frequencies for 13C’, 13Ca and 13Cb
HNCOHN(CA)CO
HN(CO)CAHNCA HN(CO)CACBHNCACB
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Collecting distance restraints for structure determination
• Distance restraints are obtained between individual protons by transfer of magnetization through thedipolar coupling
• So-called NOESY spectra are used that contain a “mixing” period where magnetization is transferredfrom one proton to another through space
• Upper distance limit: 5 Å
A NOESY correlation indicates twoprotons that are close in spaceNOE intensity ∝ r-6
N-term
C-term
C-termN-term
1H1H
Residue xResidue y
1H-1H NOESY spectrum of a protein
1H (ppm)
1H (ppm)
Distance < 5Å
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Restraint Generation
Structure Calculation
Validation
! Water
Refinement Water
Refinement Deposition
Assignment
Met24HN-N
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How about protein-ligand and protein-protein interactions?
• NMR chemical shifts are extremely sensitive to the chemical environment
• Monitor protein resonances in a 2D 1H-15N experiment (15N labeling of protein required)
• Add increasing amounts of ligand or interacting protein (unlabeled) until saturation
• Observe chemical shift perturbations (CSP) - information about interacting regions and binding affinity
kex >> CSP
• NMR can determine affinities in the milli-molar to the low micro-molar range
• NMR cannot distinguish between interactions of very high affinity
Example: Interaction of ubiquitin with an SH3 domain
J. L. Ortega-Roldan, M. Blackledge, M.R. Jensen (2018) Methods Mol. Biol. 1764: 73-85.
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Different exchange regimes give rise to different spectral features
• Fast exchange when kex >> CSP
• Kd can be determined from the variation of the CSP with ligand concentration
• Intermediate exchange when kex ≈ CSP
• CSPs are no longer a simple population-weighted average between the free and bound state chemical shifts
• The interaction kinetics (kon and koff) can be extracted
• Slow exchange when kex << CSP
• NMR spectra of the complex have to be assigned separately
• The variation of the NMR signal intensity with ligand concentration provides information about the Kd
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