Biomedical Engineering Reference
In-Depth Information
chemical shift changes of the different nuclei mapped through C9-detection
experiments provide a robust description of the interaction surface upon
formation of the Atx1-Cu(I)-Ccc2a complex.
76
On the other hand, experi-
ments that rely on DD cross-relaxation such as long-range NOESY and cross-
saturation experiments might not work efficiently when low-c nuclei are
exclusively used. Using proton polarisation as the starting magnetisation will
partially avoid the problem as one can implement the
1
H
1
H DD cross-
relaxation scheme at the beginning of the pulse sequence. Two proposals for
obtaining structural information have been described so far, exclusively using
carbon-detected experiments. One uses residual dipolar couplings,
77,78
and the
other uses paramagnetic relaxation enhancement.
79
Although, these two
techniques have been used in proton-detected experiments, there are additional
benefits to using them in carbon-detected experiments. Concerning RDC
measurements, carbon nuclei suffer less from line broadening due to their
smaller c. Thus, coupling values can be obtained precisely even for the sites
that have broad
1
H resonances.
77
In addition, PRE can be measured and is less
dependent on the local internal motion and for the sites close to the
paramagnetic centre in carbon-detected experiments. Thus, compared to
1
H
PRE,
13
C PRE is a richer source of valuable long-range distance restraints that
can define protein-protein interfaces. It is also worth noting that those
structure constraints can be obtained from high molecular weight perdeuter-
ated proteins, where only a few protons are available for structure
determination.
79
Lastly, carbon detection can also be used to extract unique
dynamics information from side-chain carbonyls.
80
This would yield valuable
information about hydrophilic protein-protein or protein-ligand interfaces
that are prone to exchange broadening and lack appropriate spins to detect.
2.7 Conclusion
Here we have discussed a variety of recently described low-c direct-detection
experiments, which take advantage of the slower relaxation properties of
nuclei. These experiments can provide complete sequence-specific assignment
of biomolecules, including protein, DNA/RNA and sugar chains. It is worth
noting that low-c direct-detection experiments are ideally suited for assigning
proline resonances. This is a clear advantage over conventional proton-
detected sequences, such as the HNCA or related experiments where prolines
cause gaps in the assignments, in particular if there are two or more in a row.
This feature is particularly important for the structural analysis of transcrip-
tion activation factors as this class of proteins often contains proline-rich
domains that are functionally important. In addition, the lower c of the nuclei
involved in the experiments is highly beneficial in the approaching metal-
binding centres of paramagnetic proteins. Furthermore, low-c direct-detection
experiments do not suffer from incomplete deuterium-to-proton back-
exchange in perdeuterated proteins that have been expressed in D
2
O. It is
particularly
important
for
large
molecular
weight
proteins
where
the
