Chemistry Reference
In-Depth Information
system to come to equilibrium [
38
]. This method differed from typical protocols
previously reported since a solution containing the NMR sample is usually used to
rehydrate the dried gel, thereby reducing sample losses from dilution. However, this
was not possible for the 4% gel, since the presence of detergent in the rehydrating
solution did not allow the original gel dimensions to be restored [
112
]. This reflects
the trade-off in mechanical stability that comes with the reduction in polyacryl-
amide concentrations required to make gels with larger pore sizes.
To address some of the difficulties in generating gel-aligned membrane protein
samples, protocols have been developed to perform the gel polymerization in a
solution already containing the protein sample, or to introduce an SDS-solubilized
sample into a gel via electrophoresis [
319
]. In the case of copolymerization, a
protocol would need to be carefully tailored to the specific features of each protein-
detergent system being studied to minimize the potential for undesired covalent
modifications during the relatively non-specific polymerization reaction [
112
]. How-
ever, a more general alternative to these neutral gels has also been developed using
charged polyacrylamide-based copolymers [
320
,
321
]. Rehydration of charged
copolymers is facilitated by a strong electroosmotic effect, providing an increase in
mechanical stability over neutral polyacrylamide gels. Consequently, alignment can
be reproducibly achieved in the presence of detergents at gel concentrations as low as
2-3%. The larger pore sizes of these lower concentration gels, along with the higher
mechanical stability, make it possible for larger protein-detergent complexes to be
introduced into the gel by rehydration. This has allowed RDC measurements to be
obtained for the 4-TM helix DsbB [
39
] and the 8-strand OmpA protein [
320
]bothin
complex with DPC micelles. In addition, alignment has been demonstrated using
copolymers with either positive or negative charge, as well as mixtures of positively
and negatively charged copolymers [
320
,
321
]. This permits the electrostatic
properties of the alignment medium to be tailored to the charge properties of the
sample, and different alignment frame orientations to be generated for the measure-
ment of more than one set of RDCs.
One aligning medium that has been extensively used for soluble proteins is
filamentous bacteriophage [
322
], which was recently been shown to be compatible
with modest concentrations (~100 mM) of phosphocholine-based membrane-
mimetics so long as the pH of the solution exceeds ~6 [
323
]. These long negatively
charged filamentous structures were also the inspiration for the design of a novel
detergent-resistant aligning medium formed by DNA nanotubes [
324
]. These are
self-assembled from concentrated solutions of a 7.3-kb “scaffold” strand mixed
with a 170 base “staple” strand, and have been used to induce alignment in the
zz
TM dimer [
324
], and the mitochondrial uncoupling protein 2 [
41
], both using
detergent concentrations in excess of 150 mM. A potentially more convenient
alternative has also been developed that generates a similar type of macromolecular
structure from the potassium salt of the dinucleotide 2
0
-deoxyguanylyl-(3
0
,5
0
)-2
0
-
deoxyguanosine [
325
]. These dinucleotides form G-tetrads that stack into columns
with dimensions similar to those of bacteriophage, and have been used for the
measurement of RDCs in the LMPG micelle-associated cytoplasmic domain of the
