1. NMR Spectroscopy – Part 2
Judith Klein-Seetharaman Department of Structural Biology

2. NMR parameters Chemical Shift H2O methyl aromatic Trp-side-chain NH OH
Backbone NH
aliphatic
Side-chain HN Ha
Spectrum see handout
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

3. 1d 1H NMR spectra
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

4. NMR of membrane proteins
In lipid bilayer:
In detergent micelle:
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

5. Problems! Detergent peaks
Detergent signals cause dynamic range problems
(Detergent signals cause spectral overlap)
Detergent deuteration is often not feasible
Problem: 1H,1H NOESY spectra do not show protein signals
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

6. Selective excitation 1H Chemical Shift [ppm] 1H Chemical Shift [ppm]
A. Selective excitation of the NH region using 90 degree pulse followed by direct observation.
B. Selective excitation of the same region as in A. Using excitation sculpting.
Backbone NH
Tryptophan side chain NH 20 15 10 5 10 5 -5
1H Chemical Shift [ppm]
1H Chemical Shift [ppm]
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

7. 2d HSQC
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Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

8. 1d projection of HSQC
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

9. HSQC spectra
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

10. Chemical shift perturbation
Figure 2 in “Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands
Laurent Volpon, Michael J Osborne, Ivan Topisirovic, Nadeem Siddiqui and Katherine LB Borden
The EMBO Journal (2006) 25, 5138–5149
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

11. Assignment is needed! 12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

12. Several different assignment strategies exist
Most easily automated:
HNCO
HNCOCACB
HNCOCA
HNCACB
HNCA
HNCACO
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

13. Experiments
Figure 9. HNCO experiment. The magnetization is transferred (blue arrows) from the HN(i) proton via the N(i) atom to the directly attached CO(i-1) carbon atom and returns the same way to the HN(i) nucleus which is directly detected. The frequencies of all three nuclei (red) are detected. Image and description downloaded from
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

14. Experiments
Figure 14. HNCA experiment. The HNCA experiment is the prototype of all triple resonance experiments. Starting at an amide proton (H) the magnetization is transferred to the directly attached nitrogen atom (N) which is measured as the first spectral dimension. Then the magnetization is transferred to the Calpha nucleus (CA) which is measured as second dimension. Afterwards, the magnetization is transferred back the same way to the amide proton which is measured as the third (direct) dimension. Image and description downloaded from
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

15. Experiments
Figure 15. HNCACO experiment. In the HN(CA)CO experiment the magnetization is transferred from the HN(i) proton via the N(i) atom and the CA nucleus (Calpha(i)) to the CO(i) carbon atom and back the same way. The Calpha atom (yellow) acts only as relay nucleus, its frequency is not detected. It is only the frequencies of HN, N and CO (red) which are detected. Image and description downloaded from
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

16. Assignments
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Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

17. Structure Prediction by NMR
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

18. NMR parameters chemical shifts NOE Dipolar coupling
Scalar coupling constants (gives dihedral angles)
Solvent exchange
HetNOE
longitudinal relaxation rates (R1)
transverse relaxation rates (R2)
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

19. The Nuclear Overhauser Effect
NMR parameters
The Nuclear Overhauser Effect
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

20. HSQC TOCSY http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

21. NMR Parameters Dipolar Couplings
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Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

22. Scalar coupling constants
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

23. Structure Calculations
Distance geometry
Determines ensembles of structures consistent with an incomplete set of distance restraints
Metric matrix algorithm
Variable target function approach
Restrained molecular dynamics
Cartesian or torsion-angle coordinate systems
Molecular dynamics force fields are supplemented by pseudo energy terms based on the NMR-derived restraints
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

24. Structure Prediction From NMR Parameters
Most widely used software suites
CNS
XPLOR
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

25. NMR parameters chemical shifts NOE Dipolar coupling
Scalar coupling constants (gives dihedral angles)
Solvent exchange
HetNOE
longitudinal relaxation rates (R1)
transverse relaxation rates (R2)
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

26. Comparison of T1 and T2 relaxation
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

27. Dynamics in folded/unfolded lysozyme
Smaller rates – more flexible
12/5/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture