Chapter 5 – Detection and Imaging Tools that Use Nonoptical Waves 191
XY plane. In this example, the 90° pulse is then applied, and the magnetization dephases
giving an FID response as before. Normally, an inversion recovery sequence is then repeated
every TR seconds to improve the signal-to-noise ratio, such that
(5.26)
S
k
T
T
T
T
I
=
−
−
−
ρ 1
2
1
1
exp
exp
R
5.4.7 MULTIDIMENSIONAL NMR
For complex biomolecules, often containing hundreds of atoms, overlapping peaks in a spec
trum obtained using just a single magnetic atomic nucleus type, the so-called 1D-NMR, can
make interpretation of the relative spatial localization of each different atom challenging.
The correct assignment of atoms for structural determination of all the major classes of
biomolecules is substantially improved by acquiring NMR spectra for one and then another
type of magnetic atomic nuclei simultaneously, known as “multidimensional NMR” or “NMR
correlation spectroscopy” (COSY). For example, the use of 2D-NMR with 13C and 15N isotopes
can be used to generate a 2D heat map plot for chemical shift for each isotope plotted on
each axis, with the 2D hotspots, as opposed to 1D peaks on their own, used to extract the
molecular signature, which is particularly useful for identify backbone structures in proteins,
a technique also referred to as “nuclear Overhauser effect spectroscopy” (NOESY). These
correlative NMR approaches can be adapted in several multichannel NMR machines for 3D-
NMR and 4D-NMR, with averaged spectra taking more like ~100 min to acquire.
Correlative NMR spectroscopy has been enormously successful in determining the
structures of several types of biomolecules. These include complex lipids, carbohydrates,
short nucleic acid sequences of ≲100 nucleotides, and peptides and proteins. The upper
molecular weight limit for proteins using these NMR methods is ~35 kDa, which is compara
tively small (e.g., an IgG antibody has a molecular weight of ~150 kDa). Multidimensional
NMR can to a great extent overcome issues of overlapping chemical shift peaks associated
with larger proteins; however, a larger issue is that the sample magnetization relaxes faster
in large proteins, which ultimately sets a limit on the time to detect the NMR signal. Larger
proteins have longer rotational correlation times and shorter transverse (T2) relaxation times,
ultimately leading to line broadening in the NMR spectrum.
Transverse relaxation optimized spectroscopy (TROSY) has been used to overcome
much of this line broadening. TROSY suppresses T2 relaxation in multidimensional NMR
spectroscopy by using constructive interference between dipole–dipole coupling and aniso
tropic chemical shifts to produce much sharper chemical shift peaks. TROSY can also be
used in combination with deuteration of larger proteins, that is, replacing 1H atoms with
2H, which further suppresses T2 relaxation. These improvements have allowed the struc
tural determination of much larger proteins and protein complexes with nucleic acids, up
to ~90 kDa.
NMR spectroscopy in its modern cutting-edge form has been used to great effect in
obtaining atomic-level structures of several important biomolecules, especially of pro
tein membranes. These are in general very difficult to crystallize, which is a requirement
of the competing atomic-level structural determination technique of x-ray crystallography.
A related spatially resolved technique used in biomedical in vivo diagnostics is magnetic res
onance imaging (MRI), discussed in Chapter 7.
5.4.8 ELECTRON SPIN RESONANCE AND ELECTRON PARAMAGNETIC RESONANCE
ESR, also referred to as EPR, relies on similar principles to NMR. However, here the reson
ance is from the absorption and emission of electromagnetic radiation due to transitions in
the spin states of the electrons as opposed to magnetic atomic nuclei. This only occurs for an
unpaired electron since paired electrons have a net spin of zero. ESR resonance peaks occur