Biomedical Engineering Reference
In-Depth Information
molecular alignment, a small residual value of the full splitting observed in the
solid state can be restored. This residual splitting carries information on the
angular orientation of a particular spin pair dipole-dipole interaction relative
to a molecular alignment tensor (see previous section).
212,213
A number of methods have been proposed to induce weak alignment of
proteins.
214-216
However, membrane proteins pose a complication as it is not
possible to use filamentous phage
214
or lipid bicelles
215
in the presence of
detergents. Polyacrylamide gels have been most widely used for membrane
protein studies. Alignment is introduced by radial or axial compression of the
gel in the NMR tube. Positively and negatively charged polyacrylamide gels
have also been developed which, combined with vertical compression, are
suitable for weak alignment of membrane proteins, and which maintain higher
stability at lower acrylamide concentrations, minimising the detrimental effect
of high acrylamide concentrations on tumbling times.
217
The magnitude of the
alignment tensor, and hence size of the RDCs, can be varied by altering the
degree of compression as well as the charge of the gel. Considered as detergent-
solubilised entities, membrane proteins are larger than comparable globular
proteins so the gel concentration or compression needs to be reduced to
prevent RDCs from becoming too big. The ability to vary the charge of the gel
also has the advantage that multiple sets of alignments can be recorded,
allowing the four-fold degeneracy of RDCs to be reduced.
217,218
Such gels were
used for alignment of the b-barrel OmpA; IPAP-HSQC and TROSY-based
HNCO experiments enabled measurement of
1
D
HN
,
1
D
C'C
a
,and
1
D
NC9
and,
when combined with distance restraints, hydrogen bonds, and chemical-shift-
based backbone dihedral angles, led to notable improvements in the accuracy
and precision of the OmpA structure.
217,219
A technical complication
surrounds transferring the protein into the gels. Polymerisation in the presence
of the protein may be detrimental due to the production of free radicals during
polymerisation. Soaking may lead to dilution due the residual water content of
gels. Gels may also be dried and then soaked in protein solution, as used for
OmpA,
217
although this may lead to some inhomogeneities in sample
preparation if the gel fails to rehydrate to its original volume.
Electrophoresis has been proposed but is not widely used.
220
Other methods
for alignment include collagen gels, polymerised in a magnetic field to induce
alignment.
221
In our experience with pSRII, the difficulty in obtaining
sufficiently high collagen concentrations results in only relatively small
RDCs. Further alignment media include DNA-based liquid crystals such as
DNA nanotubes, which consist of linear series of hexameric bundles of DNA
double helices joined end-to-end with Holliday junction crossovers and which
align in the presence of a strong magnetic field,
222,223
and G-tetrad based
structures.
224
A limitation of DNA-induced alignment is the cost of such
studies, although the use of simpler G-tetrad sequences should reduce the cost
to some extent.
224
As discussed previously, lanthanide-induced alignment can
also be used.
199
Here the rigidity of the chelating group is important to achieve
sufficient splitting.
206
