1. Lecture 8
NMR Spectroscopy
Basics and applications in biology

2. Lecture overview Basic principles of NMR spectroscopy
NMR of small molecules
NMR of proteins

3. NMR needs high magnetic fields
A good introduction into the basic principles of NMR:
For YouTube fans:

4. NMR = nuclear magnetic resonance
1H, 13C and 15N nuclei
have a very small magnetic moment: “spin 1/2”
For a single spin: two energy levels in a magnetic field
2pn = -gB0
n: frequency,
g: a constant,
B0: external magnetic field
E: energy
A spectrum always shows peaks as a function of frequency

5. More than a single spin Chemical shifts
Measured in ppm (“parts per million”) relative to a reference
Different chemical environments cause different chemical shifts
1.2 ppm
3.6 ppm D

6. More than a single spin Scalar coupling constants
Measured in Hz (“Hertz”, s-1)
Caused by different spin states of neighboring spins (“parallel”
or “antiparallel” to B0)
Between spins separated by 1, 2 or 3 chemical bonds
Doublet: 1 coupling partner
Triplet: 2 coupling partners
Quartet: 3 coupling partners D

7. NMR of urine: metabolomics
Lots of compounds detected simultaneously (“multiplexing”)
Peak integrals are directly proportional to abundance
From: Wang Y et al. PNAS 2008;105:

8. 2D NMR Two frequency axes (ppm) Often symmetrical about the diagonal
Correlates peaks in 1D NMR spectra (plotted on the sides)
Diagonal peaks
Same as 1D NMR spectrum
Cross-peaks
Connect different peaks in
1D NMR spectrum
Arise from scalar couplings
or other magnetisation
transfer mechanisms
From:

9. Metabolomics 13C-1H correlation - Greatly improved spectral resolution
H2O
13C 1H
From:

10. Protein NMR
H2O
ppm

11. Folded versus unfolded protein
Different chemical environments cause different chemical shifts

12. 2D NMR of proteins - HSQC 15N-HSQC spectrum
Correlates 15N and 1H NMR spectra
Magnetisation transfer by the scalar coupling between
amide nitrogen (15N) and amide proton (1H)
Only cross-peaks, no diagonal peaks
15N 1H

13. 2D NMR of proteins - HSQC 15N-HSQC spectrum
One peak per backbone amide
Two peaks per side-chain amide C O
15N 1H C O Cα
15N 1H R H

14. 2D NMR of proteins - HSQC
HSQC = ‘heteronuclear single-quantum coherence’
Higher magnetic field B0 improves resolution and sensitivity
Protein must be enriched with 15N
Grow E. coli on medium with 15NH4-salt as only nitrogen source
Natural abundance of 15N: 0.3%
500 MHz
950 MHz

15. Resonance assignment
Resonance assignment = attribution of a peak in the NMR
spectrum to the specific nucleus in the molecule it comes from
Needs a combination of NMR techniques
2D NOESY (NOE spectroscopy) is most important
NOESY
cross-peaks arise from nuclear Overhauser effects (NOEs)
between 1H spins
NOEs
arise from through-space dipolar interactions
provide a mechanism for magnetisation transfer
NOE intensity proportional to 1/r6 (r = internuclear distance)
observable for spins closer than ~5 Å
A NOESY cross-peak shows that two 1H spins are in close proximity

16. NOESY example NOESY Symmetrical about diagonal
Diagonal peaks correspond to 1D NMR spectrum
chentobiose

17. Protein NMR spectra NOESY
In principle sufficient information to calculate
the 3D structure of the protein

18. 3D NMR spectra
For proteins enriched with 15N and 13C

19. A bit of history Nobel prizes for NMR spectroscopy 1952 1991 2002
Felix Bloch
Edward Purcell
Richard Ernst
Kurt Wüthrich
Chemistry:
3D protein structures
by NMR
Physics: discovery of NMR
Chemistry:
FT-NMR, 2D NMR

20. and more…
2003
Peter Mansfield
Paul Lauterbur
Medicine: MR imaging

21. 3D structures of proteins by NMR
Each NOESY cross-peak presents a distance restraint
NOESY spectrum
3D structure

22. 3D structures are defined by dihedral angles
Amide bonds are planar
The backbone conformation of each amino acid residue is defined by a f and a y angle
Bond lengths and bond angles are known -> 2 degrees of freedom per amino acid backbone

23. Scalar couplings reflect dihedral angles
Karplus curve
3-bond couplings (1H-C-C-1H) depend on the dihedral angle a
Can be measured also for 1H-N-C-1H (backbone dihedral angle f)

24. NMR structures
The NMR structure of a protein is presented as a bundle of
conformers
Each conformer presents a good solution to the NMR restraints
First conformer usually is the best structure
Typically a bundle of 20 conformers is deposited in the PDB

25. Mobility NMR works in solution Can measure conformational exchange
Different experiments for different time scales

26. Drug development NMR is sensitive to changes in chemical environment
Ligand binding changes the chemical shifts
Sensitive also to weak binding
Gold standard for site-specific ligand binding
Large chem. shift changes induced by compounds 1 and 2
are highlighted in different colours
Science 1996, 274,

27. Summary I NMR owes its success to
Long life of the excited magnetisation (seconds)
Low energy ( MHz = radiofrequency)
Only nuclear spins in a magnetic field can absorb such small energy quanta
High abundance of 1H (99.985%)
Sensitivity to the chemical environment
Drawbacks of NMR
Relatively low sensitivity
Expensive magnets
Hard to become an expert

28. Summary II
NMR spectroscopy is the most versatile spectroscopy on earth
Multidimensional
Most powerful analytical tool for chemists
Metabolomics
3D structures of proteins
Mobility information
Ligand binding
MRI
NOT radioactive

29. Finally
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