using nmr spectroscopy to elucidate the structure ......collecting distance restraints for structure...

Using NMR spectroscopy to elucidate the structure,

dynamics and interactions of proteins

Malene Ringkjøbing Jensen

Institut de Biologie Structurale

Grenoble, France

malene.ringkjobing-jensen@ibs.fr

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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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60

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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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