Biomedical Engineering Reference
In-Depth Information
ordering medium 21 or in the case of nucleic acids and paramagnetic proteins,
through direct interactions with the magnetic field itself. 20,44 Alignment levels
#10 25 (i.e., corresponding to 1 in 10 5 molecules being completely aligned) lead
to RDCs that are too small compared to NMR linewidths to allow precise
measurements. Much higher degrees of alignment ($10 22 ) give rise to
extensive dipolar couplings, compromising the spectral resolution required to
analyse large biomolecules. In general, alignment levels on the order of y10 23
are optimal. 21,61 At this degree of alignment, a wide range of RDCs can be
measured with a favorable magnitude-to-precision ratio while maintaining
spectral resolution. A smaller subset of RDCs can be measured with alignment
levels y10 24
with less than optimum magnitude-to-precision ratios.
9.3.1 Ordering Media-Induced Alignment
It is now relatively straightforward to achieve alignment levels y10 23 in
solution NMR by dissolving the biomolecule of interest in an inert ordering
media 21,51,62 [Figure 9.2(A)]. This was first demonstrated using liquid crystal-
line disc-shaped phospholipids called 'bicelles' 21 which were previously used as
a mimic of membrane bilayers in studies of membrane-associated biomole-
cules. 63,64 While this neutral bicelle medium has been used in nucleic acid
studies, other media have since been introduced which have become more
popular. We provide a summary of ordering media used to date for aligning
nucleic acids in Table 9.1.
Since nucleic acids are highly negatively charged, the charge properties of
the ordering medium are an important consideration. For example, positively
charged ordering media may lead to undesirable interactions with nucleic acid
solutes. For nucleic acid applications, the ordering medium must also be
tolerant to high ionic strength conditions. The most commonly used ordering
medium that satisfies the above requirements is the commercially available
filamentous Pf1 bacteriophage, which induces alignment through electrostatic
and steric mechanisms [Figure 9.2(A)]. 65-67
Pf1 phage is composed of a 7.4 kb circular, single-stranded DNA genome
and has a rod-like shape, estimated to be y20 000 ˚ long and y60 ˚ in
diameter. 67 Pf1 phage is highly robust, having favorable properties largely due
to its lower nematic threshold. 68,69 Its coat proteins are negatively charged,
reducing the potential for adverse interactions with nucleic acids. Since
polyanionic nucleic acids have a semi-uniform charge distribution, 68,70 the
steric and electrostatic contributions from phage are thought to have similar
roles, 68,70 generally aligning nucleic acids with the principal direction of order
(S zz ) oriented along the long axis of the molecule. Positive alignment (S zz .0)
is expected for elongated nucleic acids with S zz being, on average, oriented
along the magnetic field direction [Figure 9.2(A)]. Experimentally, RDCs are
calculated from the difference in splittings measured in the absence (J)and
presence
(J + D)
of
Pf1
phage
[Figure 9.1(C)].
The
optimum
phage
concentration is typically y20 mg mL 21
but can vary depending on the
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