1. Areas of Spectrum

2. Examples of aliphatic region correlations
Ala
1.52
3.95
Thr
Asp

3. The fingerprint region – the 2nd region of interest in the COSY spectrum
Areas of Spectrum

4. COSY Fingerprint region correlating NH-aH protons

5. COSY Spectrum of a small protein
Aliphatic
Fingerprint region

6. Total correlation spectroscopy - TOCSY
Water Presaturation
Spin locking field
The spin locking field (a series of rapid 90o pulses of
varying phase) effectively averages the coupling 1H-1H
coupling constants over the entire spin system.
The dispersion of the NH-aH region allows correlations along
the entire system to become visible.

7. Even if J13 is very small, will still see transfer to it via 2
Homonuclear Hartmann-Hahn or TOCSY experiments
Under these conditions magnetisation is transferred very efficiently,
at a rate determined by J, between coupled nuclei. The longer the
mixing time, the further through the spin system the magnetisation
propagates.
J13=0.2 Hz 1 2 3
J12=7 Hz
J23=5 Hz
Even if J13 is very small, will still see transfer to it via 2

8. 8.83ppm
3.95ppm
1.52 bCH3
1.52ppm
ALA 49
Ala aH
1.52 bCH3
3.95 aH
8.83ppm

9. Connecting spin systems – The nuclear Overhauser effect (nOe)
At this point, we have used COSY and TOCSY to connect spin systems. i.e. if there are 8 arginines in the protein, we would aim to find 8 arginine patterns. Overlap or missing signals may hamper us in this initial goal. The next step is to use NOESY experiments to sequentially link the amino acid spin systems together.
The nuclear Overhauser enhancement provides data on internuclear distances. These can be more directly correlated with molecular structure.

10. W1I W1s W2 flip flip W0 flip flop W1I W1S
Consider 2 protons, I and S, not J-coupled but close in space
W1 is the normal transition probability that gives rise to a peak in the spectrum bb
W1I
W1s
W2 flip flip
W0 flip flop ba ab
W1I
W1S aa
W1 requires frequencies or magnetic field fluctuations near the
Larmor precession frequency i.e. (e.g. 500 MHz at 11.1 Tesla).
W2 requires frequencies at wI + ws, or to a good approximation, 2wI or 109 Hz
Wo is a zero quantum transition that requires frequencies at wI-ws, i.e. just the
chemical shift difference of the protons which could be 0 to a few 1000 Hz)

11. Rotational correlation time tc
In the energy level diagram for a 2 spin system, it is the transitions that involve
a simultaneous flip of both spins (cross - relaxation) that cause NOE
enhancements.
A transition corresponding to a given frequency is promoted by molecular motion
at the same frequency. Small molecules in non-viscous solvents tumble at rates
around 1011 Hz, while larger molecules such as proteins tumble at rates around
107 Hz. For small molecules, W2 will be greater than W0 and this is the dominant
mechanism for producing NOE enhancements (which turn out be positive)
For larger molecules W0 will become greater than W2 and this becomes the dominant
mechanism leading to NOE enhancements (that are now negative).
Rotational correlation time tc
rotational correlation time [in ns] is approx. equal to 0.5  molecular mass [in kDa]

12. For a small molecule, tc is small (~0
For a small molecule, tc is small (~0.3ns) and the product wtc is << 1. In this extreme narrowing limit, rotational motions include 2wo (i.e. fast motions) and W2 is preferred.
In large molecules (PROTEINS!), the tumbling is slow and wtc > 1. Wo connects energy levels of similar energy so only low frequencies are required. Therefore this is the preferred mechanism in large molecules. It is known as cross-relaxation.
In the 2D NOESY experiment, an additional mixing time is added to the basic COSY sequence. The result is a build up of magnetisation from one nucleus to a close neighbour.
90o
90o
90o t2 t1
Mixing time
Presat
(magnetisation components of interest lie along –z). Cross relaxation now occurs to nearby nuclei.

13. The NOE operates ‘through space’, it does not require the nuclei to be chemically bonded. The build-up is proportional to the separation of the two nuclei
nuclear separation
If we calibrate this function by measuring a known distance in the
protein and the intensity of the NOE, we can write
where k is a
proportionality
constant

14. The power of the NOESY experiment is that the intensity of an
NOE peak will be related to the nuclear separation.
Strong NOE crosspeaks Å
Weak NOE crosspeaks Å
Extending the mixing time will permit nuclei separated by 5Å - not
all spin systems will give a detectable peak though. So the absence of
a peak does not preclude close approach. Similarly a weaker
crosspeak does not always prove a larger internuclear distance.
Therefore tend to be cautious and define distance ranges.
Strong ( Å), medium ( Å), weak ( Å).
Since this works through space we can use the NOE to connect spin
systems that we assigned with the COSY and TOCSY spectra.

15. Sequential ‘walking’ with sequential nOes1 2 3 4 5
Fingerprint region
of a 2D NOESY dH
TOCSY gH bH
COSY NH
9.0
8.0
7.0
NOE
COSY
NOE
TOCSY
NOE
Ala

16. NH-NH Contacts dH NOE gH bH Ala 9.0 8.0 7.01 2 3 4 5 dH
NOE gH bH
Ala
9.0
8.0
7.0
The ‘NH-NH’ region provides an
additional source of sequential
contacts - note the symmetry
around the diagonal and that
this contact does not give direction.

17. aHi-NHi+3
aHi-NHi+1

18. An a-helix can be recognised by repeating patterns of short
range nOes. A short range nOe
is defined as a contact between
residues less than five apart in
the sequence (sequential nOes
connect neighbouring residues)
For an a-helix we see aHi-NHi+3
and aHi-NHi+4 nOes.
i+4
i+3 N H H
NOE H
i+2 i

19. A b-strand is distinguished by strong CaHi-NHi+1contacts and long range nOes connecting the strands.
A long range nOe connects residues more than 5 residues apart in the chain.

20. Assignment of secondary structural segments
sequential stretches of residues with consistent secondary structure characteristics provide a reliable indication of the location of these structural segments