Biomedical Engineering Reference
In-Depth Information
translational diffusion rates for pSRII in q 5 0.4 and q 5 0.25 DMPC/c7-
DHPC bicelles at 35 uC are shown in Figure 12.3. Assignments were
transferred from spectra in c7-DHPC micelles achieving 58% and 81%
assignment respectively. In solid-state NMR, larger bicelles, typically with q
.3 are used. Although bicelles are envisaged as a disc of lipids, it is likely that
a variety of shapes and sizes of particle exist, meaning that their homogeneity
is unlikely to be as high as for nanodiscs; in a recent AFM study, large discs,
small discs and worm-like structures were identified. The small bicelle discs
were estimated to have a diameter of ca. 8.7-9.6 nm.
152
Since the lipid and
bounding detergent are in different chemical environments,
31
P and
1
H1D
NMR experiments can identify the different species. In contrast, in mixtures of
phospholipids and detergents it is found that these resonances overlap.
152,153
This allows easy verification of the correct formation of the bicelles, as well as
the lipid-to-detergent ratio attained.
Bicelles have enabled structural studies by NMR in a variety of contexts:
protein-protein interactions forming the a
IIb
b
2
integrin dimer which are
broken in harsher micelle systems are found to persist in bicelles.
154
Structure
determination of this complex using solution NMR was achieved in c6-DHPC,
palmitoyl-oleoylphosphatidylcholine, palmitoyl-oleoylphosphatidylserine
bicelles and in addition it was possible to characterise slow (ms) timescale
dynamics between the monomeric a
IIb
and b
2
subunits and the dimeric integrin
form.
154
The small multidrug resistance protein (Smr) can be stabilised in its
functional form in DMPC/c6-DHPC bicelles with q 5 0.33.
155,156
The protein
was shown to be in its native dimeric form and was found to be functional
based on ligand-binding assays, resulting in backbone assignment; sample
lifetimes could be extended by employing ether-linked DMPC and DHPC
analogues.
155-158
High-quality 2D [
1
H,
15
N] TROSY spectra of the large b-
barrel membrane protein OmpX in DMPC/c6-DHPC bicelles demonstrate the
suitability of this medium for structural studies of larger systems.
153
NOE data
for this system demonstrate that OmpX is only in contact with the lipid tails,
supporting the use of this system as a suitable membrane mimic.
153
The ability to alter the size of the bicelles by varying the concentration of
one of the components may allow the combination of restraints from both
solution and solid-state NMR studies. This approach has already been used to
obtain restraints from membrane proteins in micelles and lipid bilayers;
159,160
however, varying the 'q-value' of bicelles would enable an identical medium to
be used in both cases.
161
The concept of carrying out a 'q-titration' has also
been used to identify variations in dynamics.
162,163
12.4 Isotope-Labelling Strategies
One of the main difficulties in studying membrane proteins is that the
molecular size of the protein under study is increased by the presence of the
membrane mimic. This leads to broader lines and lower sensitivity when
compared to studies of water-soluble proteins of an equivalent size. Amino
