A presentation made for 2nd year Biochemistry undergrads to learn about the NMR structure determination process.

1. NMR TUTORIAL 1I
PROTEIN NMR
... from data to structure.
for 2nd year Biochemists
by Christiane Riedinger

2. why do we solve NMR structures ?
• ... can’t get a crystal structure :-)
• study the protein in solution
• obtain information about dynamics
in general:
• infer function from structure
• map interaction sites of ligands
• molecular mechanisms
C.Riedinger 2009

3. The process of NMR structure determination
1. the sample
2. sequential assignment
3. side-chain assignment
4. collection of NMR restraints
5. NMR structure calculation
6. structure validation
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4. 1. The Sample
• protein of interest (now up to ~60kDa), ideally well folded
• high concentration (mM), no aggregation/precipitation, stable over time
• isotopically labelled, 15N, 13C, 2H ...
• keep salt concentration low, more signal
• recent advances for larger / less soluble / less well folded proteins:
decrease overlap - multi-dimensional spectra
high MW - TROSY effect
improve s/n - cryogenic probe-heads
C.Riedinger 2009

5. 2. NMR assignments (general)
example protein
• we want to know which atom
each peak in our spectra
correspond to (that’s MANY!!)
• 1st carry out sequential assignment
know the sequence!
(obtain chemical shift of backbone amides)
• then side-chain assignment
(obtain chemical shift of all other atoms)
C.Riedinger 2009

6. 2. sequential
assignments
(side-chains
with amides)
•1H/15N HSQC spectrum: one
peak for each backbone amide
(and side-chains containing amides)
• distribution of peaks depends on
chemical environment of amide in
the protein
• aim: assign each peak in HSQC
to an amide in the primary
sequence of your protein.
• in order you achieve this, a set of
3D experiments are acquired... protein sequence
C.Riedinger 2009

7. concept of a 3D
spectrum:
• label HN frequencies with the frequency of a third nucleus, for example side-chain carbons
• “spread out” HSQC along a third dimension
• (of course there are other 3D spectra too, which are not HN based...)
C.Riedinger 2009

8. concept of a 3D
spectrum:
15N
1H
• label HN frequencies with the frequency of a third nucleus, for example side-chain carbons
• “spread out” HSQC along a third dimension
• (of course there are other 3D spectra too, which are not HN based...)
C.Riedinger 2009

9. concept of a 3D
spectrum:
15N
C
13
1H
• label HN frequencies with the frequency of a third nucleus, for example side-chain carbons
• “spread out” HSQC along a third dimension
• (of course there are other 3D spectra too, which are not HN based...)
C.Riedinger 2009

10. concept of a 3D
spectrum:
15N
C
13
1H
• label HN frequencies with the frequency of a third nucleus, for example side-chain carbons
• “spread out” HSQC along a third dimension
• (of course there are other 3D spectra too, which are not HN based...)
C.Riedinger 2009

11. generally, two types of spectra are collected:
e.g. CBCACONH e.g. CBCANH
links HN frequencies with Ca and Cb links HN frequencies with Ca and Cb
chemical shifts of previous residue (i-1) chemical shifts of previous residue (i-1)
in sequence in sequence and those of itself (i)
C.Riedinger 2009

12. generally, two types of spectra are collected:
Spectrum A:
IDENTIFIES amino acid
e.g. CBCACONH e.g. CBCANH
i-1 i
links HN frequencies with Ca and Cb links HN frequencies with Ca and Cb
chemical shifts of previous residue (i-1) chemical shifts of previous residue (i-1)
in sequence in sequence and those of itself (i)
C.Riedinger 2009

13. generally, two types of spectra are collected:
Spectrum A: Spectrum B:
IDENTIFIES amino acid CONNECTS amino acid
to its neighbours in the
sequence
e.g. CBCACONH e.g. CBCANH
β
α
i-1 i i-1 i
links HN frequencies with Ca and Cb links HN frequencies with Ca and Cb
chemical shifts of previous residue (i-1) chemical shifts of previous residue (i-1)
in sequence in sequence and those of itself (i)
C.Riedinger 2009

14. Analysing the CBCACONH and CBCANH
• look along the carbon dimensions for each peak in the
HSQC in both 3D spectra (this is called “strip plot”)
“strip plot”
• for the CBCACONH (A) you will see two peaks, the Ca and
Cb of the previous residue in the sequence
Cb (i)
• for the CBCANH (B) you will ideally see four peaks, the Ca
and Cb of the previous residue as well as those of itself
Cb (i-1)
• by comparing the two strips, you can identify which peaks
belong to the previous residue (i-1) or to itself (i).
• from the chemical shifts (=the position of the peaks), you
Ca (i-1) can take a guess as to which amino acids you are dealing
with.
Ca (i)
A B
13C
15N
13C
C.Riedinger 2009 1H

15. Analysing the CBCACONH and CBCANH
• look along the carbon dimensions for each peak in the
HSQC in both 3D spectra (this is called “strip plot”)
“strip plot”
• for the CBCACONH (A) you will see two peaks, the Ca and
Cb of the previous residue in the sequence
Cb (i)
• for the CBCANH (B) you will ideally see four peaks, the Ca
and Cb of the previous residue as well as those of itself
Cb (i-1)
• by comparing the two strips, you can identify which peaks
belong to the previous residue (i-1) or to itself (i).
• from the chemical shifts (=the position of the peaks), you
Ca (i-1) can take a guess as to which amino acids you are dealing
with.
Ca (i)
A B
13C
15N
13C
C.Riedinger 2009 1H

16. Identifying Amino Acids from Chemical Shifts
• due to their chemical structure, side-chain atoms of amino acids have dispersed chemical
shifts
• for a complete list of average chemical shifts, see http://www.bmrb.wisc.edu/ref_info/
statful.htm
• based on Ca and Cb chemical shifts alone, some amino acids such as alanines, serines/
threonines and glycines can be unambigously identified, others can be at least narrowed
down to a few possibilities:
C.Riedinger 2009

17. Placing strips in the sequence...
13C
A B A B A B A B A B
13C
• now you compare the strips of different amide peaks with each other
• for the i-peaks in the CBCANH, you should find matching peaks in a different strip of the CBCACONH
• for the i-1 peaks in the CBCACONH, you should find matching i-peaks in a different strip of the CBCANH
• remember: the chemical shifts of side-chain atoms of a residue X will be identical, whether they are “seen”
from the position of the previous amide or itself!
• now compare a chain of strips and their possible amino acid types to the primary sequence. That’s it!
C.Riedinger 2009

18. Placing strips in the sequence...
13C
A B A B A B A B A B
13C
• now you compare the strips of different amide peaks with each other
• for the i-peaks in the CBCANH, you should find matching peaks in a different strip of the CBCACONH
• for the i-1 peaks in the CBCACONH, you should find matching i-peaks in a different strip of the CBCANH
• remember: the chemical shifts of side-chain atoms of a residue X will be identical, whether they are “seen”
from the position of the previous amide or itself!
• now compare a chain of strips and their possible amino acid types to the primary sequence. That’s it!
C.Riedinger 2009

19. Real data...
In reality it’s not as pretty!
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20. 3. side-chain assignments
• after completing the backbone assignments, you proceed to the side-chain assignments, i.e.
the remaining carbon atoms and hydrogen atoms of your protein’s side-chains
• for the HN-based experiments, you might again analyse the data in a strip plot
• you can identify the different side-chain hydrogen atoms based on their chemical shift:
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21. • here is a list of experiments you might acquire (of course there are many more!):
• note: not all of them are HN based, these spectra are analysed by starting with a carbon-
hydrogen pair with known chemical shifts
• for example, if you know the chemical shifts for the Ca and Ha of a particular residue X, you
move to this position in the HCCH-COSY and collect the chemical shifts for the adjacent
hydrogens in the side-chain
• use the carbon HSQC to collect any final missing assignments, but also to obtain the most
precise chemical shift, as this spectrum will have the highest resolution (it’s just a 2D)
C.Riedinger 2009

22. side-chain
assignments
The carbon HSQC shows
all side-chain carbon-
hydrogen pairs in one
spectrum.
Going through all your data,
you will eventually obtain a full
list of chemical shifts for all
assignable atoms in your protein. 1H/13C
HSQC
C.Riedinger 2009

23. 4. NMR restraints: Define the structure
We usually obtain the following structural restrains from NMR data:
1. the NOE (measures distance between two atoms)
2. torsion angles (define the rotation around bonds)
3. Residual dipolar couplings (RDCs)
(provide global, orientational restraints)
C.Riedinger 2009

24. 1. the NOE (nuclear Overhauser effect)
• is strictly local phenomenon!!!
• measure a change in intensity of one resonance when neighbouring
nucleus is perturbed
identify peaks
• distance dependent! NOE~1/r6 assign to proton-pair
• spins that are less than 5A apart calibrate to distance
• intensity of cross-peaks ~ separation of the nuclei
• translate peak intensity into distance! (calibration required)
• separation ranges: 1.8-2.7A, 1.8-3.3A, 1.8-5A (strong, medium, weak)
• lower bound = VDW radius
C.Riedinger 2009

25. 2. Torsion angles
• torsion angles, dihedral angle (the angle defined between two planes)
• phi, psi and omega(=180*)
• define the orientation of the backbone!
• torsion angles are related to J-couplings through the Karplus equation
• J-couplings = scalar through-bond couplings ... formation of multiplets
• measure in Hz
• or predict from carbon chemical shifts! (TALOS, part of NMRPipe)
C.Riedinger 2009

26. 2. Torsion angles
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27. 3. Residual Dipolar Couplings
decoupled,
isotropic
J coupling,
isotropic
• global restraints!!
• interaction between two magnetic dipoles
• the DC depends on the distance between splitting = J-coupling (Hz)
two nuclei (r) and the angle of the bond
J-coupling plus RDC!
(psi) relative to the magnetic field (B0) using alignment medium
• measure the orientation of a bond with
respect to magnetic field! splitting = J-coupling + RDC (Hz)
C.Riedinger 2009

28. C.Riedinger 2009

29. 5. NMR Structure Calculation
- experimental restraints:
NOEs, torsion angles, RDCs
- other known parameters:
VdW radii, bond angles
= “force field” / “target function”
- describes potential energy
- 3D energy landscape
- impossible to enumerate all solns!
- aim of structure calculation
determine the energetic minimum
that combines the empirical force
field with experimental restraints.
- minimise!!!!

30. Minimisation of the Target Function
• Cartesian space or torsion angle space
• t.a.s: only rotation around angles, bond lengths not affected
• molecular dynamics: overcome local energy barriers with Ekin
• = simulate elevated temperature!
• “Simulated annealing”
• start with high Ekin (maximise sampling of conformational space)
• then cool down
• generate a number of models that are in agreement with the
experimental data
Note: this picture was obtained from “the internet”
a few years ago, so unfortunately, there is no
C.Riedinger 2009 reference. All other figures have been made by me.

31. The result
PDB code:
1Z1M
C.Riedinger 2009

32. 5. The work is not done...
Refinement...
Validation...
• some structure calculations use simplified force-fields that introduce errors that need to
be fixed afterwards
• a very useful structure validation tool is part of the “WhatIf” molecular modelling
package: http://swift.cmbi.ru.nl/servers/html/index.html
Simply submit a PDB file and the software will check for most common errors in
nomenclature, packing, planarity, torsion angles...
• try your favourite structure of the PDB database and see how good it is!
C.Riedinger 2009