NUCLEAR MAGNETIC RESONANCE 115
(NOESY) experiment must be used. See Section 3.4.10. The NOESY experi-
ment transfers magnetization through space, and, therefore, it will show cross
peaks to all protons that are close in space (within 4 – 5 Å of each other) regard-
less of whether they are in the same spin system or not. In a folded protein,
aa residues are inherently close in space, so assignments can be made by com-
paring the peaks in the NOESY experiment with other spin systems deter-
mined by COSY and TOCSY.
3.4.10 Nuclear Overhauser Effect Spectroscopy (NOESY)
NOESY NMR spectroscopy is a homonuclear two - dimensional experiment
that identifi es proton nuclei that are close to each other in space. If one has
already identifi ed proton resonances in one - dimensional NMR spectroscopy
or by other methods, it is then possible to determine three - dimensional struc-
ture through NOESY. For instance, it is possible to determine how large mol-
ecules such as proteins fold themselves in three - dimensional space using the
NOESY technique. The solution structures thus determined can be compared
with solid - state information on the same protein obtained from X - ray crystal-
lographic studies. The pulse sequence for a simple NOESY experiment is
shown in Figure 3.17.
The fi rst 90 ° pulse places magnetization in the xy plane, and the two com-
ponents then precess at their individual frequencies fort 1. At the end of t 1 ,
each component will have its ownx and y component of magnetization. The
second 90 ° pulse places the magnetization into the xz plane, where the y com-
ponents continue to precess about thez axis. A short mixing time, τm (typically
0.05 s), follows, allowing an exchange of magnetization to take place. At the
end ofτm the two component frequencies have been modulated through the
process of exchange going on between the nuclei (this in turn is a function of
t 1 ). An FID is produced by a third 90 ° pulse, which moves the magnetization
back into thexy plane. Transformation of the FID stacks yields the two -
dimensional trace with cross peaks between exchanging nuclei. The pulse
sequence is set up to detect two uncoupled spins having different chemical
shifts but that undergo slow exchange. In nonexchanging nuclei, through - space
Figure 3.17 A pulse sequence used for a NOESY NMR experiment. (Adapted with
permission of Nelson Thornes Ltd. from Figure 8.12 of reference 21 .)
90° pulse
(x)90° pulse
(x)90° pulse
(x)
t 1 τm t 2FID