1 Basic principles
1. What particles are magnetic? May a magnetic particle have a zero spin
number? May a magnetic particle have a zero electric charge? Give examples
of nuclei which are magnetic. What nuclei are found in biomacromolecules?
Which of them are magnetic? Which of them can be routinely
studied by NMR?
2. What is the relation between energy diﬀerence of stationary spin states
and the external magnetic ﬁeld. How is the frequency of precession related
to the external magnetic ﬁeld? What determines how many nuclei are in
the individual states?
3. The precession frequency of protons in diﬀerent chemical groups diﬀer.
Why? Give a relative size of these deviations (in orders of magnitude).
4. Which of the following interactions change the average precession frequency
of nuclei in molecules dissolved in an isotropic solvents and which of
them signiﬁcantly contribute to relaxation: direct interaction of magnetic
dipolar moments, shielding by electrons, interaction of magnetic dipolar
moments mediated by electrons of chemical bonds connecting the interacting
atoms.
5. Is the presence of a static homogeneous external magnetic ﬁeld necessary
to create the macroscopic magnetization? Is it suﬃcient to have the static
homogeneous external magnetic ﬁeld in order to create the macroscopic
magnetization? Is it suﬃcient to have the static homogeneous external
magnetic ﬁeld in order to observe signal in an NMR spectrometer? If not,
what else is needed?
6. Explain the following terms: ninety-degree pulse, carrier frequency, frequency
oﬀset.
7. What is the Fourier transform good for in NMR spectroscopy?
8. Explain the idea of a two dimensional experiment.
9. Describe evolution of chemical shift and scalar coupling during various
echoes.
10. Describe INEPT.
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2 NMR spectroscopy of nucleic acids
11. Using Fig.1, explain the terms purine base, pyrimidine base, nucleotide,
and nucleoside. Which of the atoms are involved in forming hydrogen
bonds in Watson-Crick and Hoogsteen base pairing?
12. In Fig. 1, show numbering of purine and pyrimidine bases and dihedral
angles α, β, γ, δ, , ζ, and χ. Explain the concept of pseudorotation angle
for describing sugar conformations.
13. Draw the structure formulas of Adenine, Cytosine, Guanine, Thymine and
Uracil. Which of the hydrogen atoms are in the fast exchange regime with
solvent water? How does the exchange aﬀect the measured NMR spectra?
14. Which quick NMR experiment would you use for a newly prepared oligonucleotide
sample to check whether it is suitable for further study by NMR?
Which of the following solvents would you use to prepare the sample and
why? (H2O pure, D2O pure, mixture 90% H2O and 10% D2O).
15. Explain the syn- and anti conformations around the glycosidic bond. How
would you use NMR spectroscopy to distinguish between these two con-
formations?
16. What is sugar puckering? Which sugar conformations are prevalent in
DNA and RNA? How can you study the sugar conformation by NMR
spectroscopy?
17. Is the spectrum in Fig. 2 COSY or NOESY spectrum? What kind of
interaction gives rise to the cross-peaks in this type of spectra? Is the
spectrum in Fig. 2 one of a DNA or RNA? How many nucleosides of each
kind does the molecule include provided the sample is a symmetric duplex
and all the bases form Watson-Crick base pairs?
18. Using Fig. 3–5, explain the principle of sequential assignment in nucleic
acids using NOESY spectra.
19. Explain the diﬀerence between the assignment of nucleic acids using throughspace
(NOESY) and through-bond interactions. Which of the methods is
more reliable and why? What kind of nucleic acid sample do you need for
the through-bond triple-resonance experiments to be applicable?
20. Name the most important heteronuclear experiments used for the assignment
of nucleic acids and explain their use.
2
3 NMR relaxation
21. Describe two most important physical mechanisms of NMR relaxation (for
spin-1/2 nuclei). Describe their contributions to the R1 and R2 relaxation
rates.
22. Describe the terms ”correlation function” and ”correlation time” for a
rigid spherical molecule in the absence of chemical/conformational changes
modifying the chemical shift tensor. Use the loss of coherence as an ex-
ample.
23. How is NMR relaxation related to molecular motions? Describe the term
”spectral density function”. Describe the eﬀect of ﬂuctuating ﬁelds on
the loss of coherence and on the return of the total magnetization to its
equilibrium value.
24. What is the eﬀect of slow (slower than the correlation time of the overall
tumbling) chemical/conformational changes modifying the chemical shift
tensor on NMR relaxation? How are the relaxation rates R1 and R2
inﬂuenced?
25. What is the eﬀect of internal motions of biomolecules on relaxation of
their NMR signals? Use an example of relaxation of 15
N in amide groups
in the protein backbone and assume that the internal motions and the
overall tumbling are independent.
26. Describe the principles of measuring relaxation rates in biomolecules.
What relaxation parameters are usually measured in practice?
27. What methods can be used to interpret NMR relaxation parameters in
the terms of intramolecular motions? Explain the basic principles.
28. Describe the procedure for the model-free analysis of relaxation data.
29. On what assumptions is the model-free analysis based?
30. Describe the procedure of the spectral density mapping. Under what
conditions is the reduced spectral density mapping applicable?
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Extra questions for those who did not attend/pass
the practical course C6775:
4 Assignment of protein NMR spectra
1. Draw examples of individual 1
H, 15
N, and 13
C spin systems in a protein
chain.
2. How many peaks should appear in a well-resolved 1
H-15
N HSQC spectrum
of a peptide MTHLNKWPEQ?
3. How many peaks should appear in a well-resolved 1
H-15
N HSQC spectrum
of a peptide MYPCTGQNLE?
4. How many peaks should appear in a well-resolved 1
H-15
N HSQC spectrum
of a peptide MIGPWLKNVD?
5. With the help of the table of typical carbon chemical shifts, assign the
following chemical shifts (obtained from an HNCACB experiment) to alanine,
tyrosine, tryptophan, glycine, and threonine: (62.3 ppm and 68.4 ppm),
(50.3 ppm and 18.4 ppm), (56.3 ppm and 39.4 ppm), (42.3 ppm), and (60.3 ppm
and 29.4 ppm).
6. With the help of the table of typical carbon chemical shifts, assign the
following chemical shifts (obtained from an HNCACB experiment) to alanine,
isoleucine, histidine, glycine, and serine: (62.3 ppm and 38.4 ppm),
(53.3 ppm and 28.4 ppm), (50.3 ppm and 19.4 ppm), (44.3 ppm), and (60.3 ppm
and 57.4 ppm).
7. Identify amino acid which showed the following 1
H/13
C chemical shifts (in
ppm) in the HCCH-TOCSY spectrum: 4.23/62.1, 2.01/31.9, 0.63/20.4,
and 0.81/21.9.
8. Identify amino acid which showed the following 1
H/13
C chemical shifts (in
ppm) in the HCCH-TOCSY spectrum: 4.93/59.1, 4.35/62.9, and 4.13/62.9.
9. Identify amino acid which showed the following 1
H/13
C chemical shifts (in
ppm) in the HCCH-TOCSY spectrum: 4.53/62.1, 5.35/69.9, and 1.23/22.9.
10. Identify amino acid which showed the following 1
H/13
C chemical shifts (in
ppm) in the HCCH-TOCSY spectrum: 4.23/60.1, 3.35/41.9, 3.25/41.9,
2.00/30.7, 1.85/30.7, 1.92/28.4 and 1.63/28.4.
4
5 NMR structure determination
11. Describe diﬀerence between chemical conformation and conﬁguration of
biomacromolecules, their relative importance in the structure determination
of small and large molecules, role of torsion angles.
12. Describe how the three-bond scalar coupling constant between amide and
alpha proton reﬂect secondary structure of the protein.
13. Brieﬂy explain at least one method of the three-bond scalar coupling mea-
surement.
14. What inter-proton distances serve as good indicators of β-sheets and helical
structures. Is it possible to distinguish α and 310 helix based on NOE
cross-peaks (if so, how?)?
15. How can one predict secondary structure just from chemical shift values?
16. What structural information can be obtained from residual dipolar couplings
and how is the residual dipolar coupling measurement done in prac-
tice?
17. It is not possible to calculate three-dimensional structure from the measured
distances by simply solving the trigonometric equations (distance
geometry). Explain why.
18. Compare advantages and disadvantages of using diﬀerent structure-related
types of NMR data (NOE, scalar couplings, dipolar couplings) in the structure
determination.
19. Brieﬂy describe the principles of molecular dynamics simulations. Why
do we use experimental data when computational methods are available.
20. Explain how NMR data are introduced into the structure calculations
using molecular dynamics.
5
N
O
O
OH
O
O
-
OP
N
O
OH
N
NH
N
NH2
O
N
NH2
O5'
Figure 1:
6
Figure 2:
7
Figure 3:
8
Figure 4:
9
Figure 5:
10