display broad lines and the poor resolution limit imposes a fundamental
limitation. In the new approaches, such as transverse relaxation-optimized
spectroscopy, or TROSY, the pulse sequences are designed to suppress harm-
ful spin-relaxation effects that cause decay of the NMR signals. Among
the scientists who developed these techniques was Kurt Würthrich, who
was awarded a Nobel Prize in 2002 in recognition of his efforts.
The analysis of NMR spectra is often limited by the width of the indi-
vidual peaks. One major source of linewidth is anisotropy of the chemical
shift. In solution, if the protein is tumbling rapidly only the average value
is observed. However, if the motion is slow, then the entire anisotropy
contributes and the lines are broad. Such reduced motion is a problem
with large proteins or proteins that aggregate or form large complexes.
Special pulse techniques can be used to reduce linewidths. For example,
the dipolar fields of protons may be reduced by the use of so-called
decoupling procedures. The use of TROSY partially alleviates the effect
of relaxation, which is suppressed by adjusting the pulse sequence to
create field strengths where the transverse relaxation rate is minimal. A
comparison of the spectra from TROSY and the more conventional COSY
is shown (Figure 16.10). In addition, these efforts have benefited from
new labeling strategies.
As is evident in the TROSY spectra, NMR spectra are commonly obtained
with the use of two different types of nuclear spin, such as protons and(^15) N or (^13) C. For proteins isolated from expression systems, the protein
can be labeled with these isotopes using enriched media. For^15 N, the
naturalabundance of the isotope is sufficient, although the protein con-
centration must be poised sufficiently, high. In such a heteronuclear
system, the spectrum contains a peak for each unique proton associated
with the nonproton nuclear spin. One of the most common experiments
for proteins is to measure the heteronuclear single quantum correlation
(HSQC) spectrumusing^15 N. Every amino acid residue, excluding proline,
has an amide proton attached to nitrogen in the peptide bond spin in
addition to any sidechains that contain a nitrogen-bound proton. Thus
the HSQC spectrum ideally provides a straightforward approach for
identifying a contribution for every residue, provided that the protein
is folded properly.
As an example, TROSY experiments have been performed on nucleic
acids to provide direct experimental evidence for hydrogen-bonding inter-
actions, which are usually only inferred from X-ray structures. These experi-
ments made use of^15 N, which has a nuclear spin of 1/2, substituted for
the predominant isotope^14 N, which has no nuclear spin (Table 16.1). For
DNA, the^15 N–^15 N couplings across Watson–Crick hydrogen bonds can
be identified in oligonucleotides while simultaneously monitoring the^1 H
couplings (Figure 16.11). These experiments are intrinsically low-sensitivity
measurements and therefore require the use of the better-resolution TROSY
approach. While TROSY is useful in enhancing the resolution of amide