Biomedical Engineering Reference
In-Depth Information
In practice, the requirement for measuring splittings at multiple fields
decreases the number of RDCs that can be measured reliably given the
decrease in spectral resolution at lower field strengths. Recently, Bax and co-
workers showed that an approximate value for the scalar imino N-H scalar
coupling (J
NH
) in base-paired residues can be obtained based on the imino
proton chemical shift, allowing the reliable measurement of field-induced
RDCs at a single magnetic field strength.
77
Another important consideration when measuring field-induced RDCs is
that splittings have a field-dependent contribution from dynamic frequency
shifts (DFS), which arise from the imaginary component of the spectral density
function for cross-correlation between dipolar and chemical shift anisotropy
relaxation mechanisms.
83,84
However, at fields .500 MHz, the DFS
contribution to splittings is nearly constant (within 0.1 Hz), resulting in a
relatively small contribution to the measured RDCs (typically ,0.2 Hz for C-
H and N-H RDCs measured at fields $500 MHz).
Even at current magnetic field strengths, the achievable degree of magnetic
field alignment (10
24
) for typical RNA constructs (20-40 nucleotides) is still an
order of magnitude smaller than the optimal degree of alignment (10
23
).
However, field alignment can allow measurement of an independent set of RDC
data without having to use a potentially perturbing ordering medium.
81
The
overall x-tensor also has a relatively simple and well-known dependence on
structure, and in particular, the orientation of nucleobases. This makes possible
a number of unique applications such as the determination of nucleic acid
stoichiometry,
81
derivation of the relative orientation of nucleic acid-protein
complexes when the nucleic acid structure is known,
52,76
and determination of
the absolute levels of internal dynamics.
85,86
Note that such applications often
require accurate parameters for the nucleobase x-tensors, and any uncertainty in
these parameters need to be properly accounted for.
87
For larger and extended
RNA molecules, more optimal levels of alignment may be achievable,
particularly as the alignment grows quadratically with the ever-increasing
magnetic field strength. For example, optimal alignment levels of 10
23
are in
principle achievable at current field strength (900 MHz) for RNA on the order of
100 base pairs, and much larger RNA molecules can now be studied by NMR
spectroscopy.
23
We therefore anticipate that field RDCs will continue to be
important parameters in NMR studies of RNA structure and dynamics.
9.4 Measurement of RDCs in Nucleic Acids
Several experiments have been developed to measure a wide variety of RDCs
in nucleic acids (see Table 9.2). The choice of RDCs to be measured is
generally guided by the desire to maximise the magnitude-to-precision ratio
and coverage of data throughout the RNA base, sugar and backbone moieties.
The most commonly and easily measured RDCs are those between directly
bonded C-H, N-H, and C-C nuclei in the nucleobases and also C19-H19 in the
sugar moieties (Figure 9.3). For small RNA molecules, these directly bonded
