Chemistry Reference
In-Depth Information
lyophilized (e.g., BNip3 [
188
], EphA1 [
189
], integrin
3[
184
]) or acetone-
precipitated (the potassium channel voltage sensing domain [
153
]) states, allowing
direct resolubilization into bicelle solutions. Similarly, proteins that are purified
into organic solvents can be directly added to an organic solution of bicelle-forming
lipids. As shown for Smr, evaporation of the organic solvent followed by resuspen-
sion in aqueous buffer can generate a functional bicelle-solubilized receptor [
183
].
For proteins that cannot be refolded directly into bicelle solutions, it may be
possible to transfer a micelle-solubilized sample into a bicelle solution through on-
column exchange, as was done for KCNE3 [
190
]. Alternatively, for proteins that
cannot be folded in bicelle-compatible micelles, it may be necessary first to
reconstitute into lipid vesicles [
186
]. Once in this state, sample concentration and
buffer exchange can be achieved by centrifugation, followed by resuspension of the
pellet in the bicelle-forming detergent solution. This approach was found to be
required for the incorporation of the G protein-coupled receptor CXCR1 into small
bicelles [
191
]. However, only extramembraneous N- and C-terminal regions of the
sample could be observed in
1
H-
15
N HSQC spectra, and only for very low
q
ratio
bicelles (0.1).
When working with bicelles, the stability of the bicelle itself may require special
attention since hydrolysis of lipid carboxy-ester bonds changes the composition of the
bicelle over time, leading to phase separation [
192
]. This reaction is accelerated under
acidic or basic conditions, with the more water-accessible state of the short-chain
phase being particularly susceptible to hydrolysis. For this reason, the hydrolysis-
resistant ether-linked analog 6-
O
-PC (1,2-di-
O
-hexyl-
sn
-glycero-3-phosphocholine)
is being increasingly used to extend bicelle sample lifetimes [
186
]. It is also possible to
use ether-linked long-chain lipids (e.g., 14-
O
-PC or 16-
O
-PC), although this change in
the lipid headgroup can alter the structure of embedded proteins [
193
]. Bicelle stability
can also be improved by spiking the solution with charged amphiphiles to increase
charge repulsion between bicelles [
194
,
195
]. Both stabilization strategies were used
for Smr, with a 3:1 mixture of short- and long-chain ether lipids (6-
O
-PC and 14-
O
-
PC), doped with 10% of a 3:1 mixture of short- and long-chain phosphatidylserines
[
183
]. Even with all these modifications to the basic bicelle mix, the sample half-life
was typically on the order of ~1 week, illustrating the inherent difficulties of
maintaining some membrane proteins in a folded functional state.
One aspect of any bicelle solution that should not be neglected is the significant
concentration of monomeric detergent that exists in equilibrium with the bicelle-
bound state. These small amphipathic molecules may be capable of binding to any
exposed hydrophobic patches on the protein that protrude from the bicelle [
196
],
potentially destabilizing the protein [
165
]. Under these circumstances the slightly
larger hydrophobicity of ether lipids may confer an additional advantage since
this gives rise to a small decrease in monomeric detergent concentrations [
197
].
More dramatic reductions in monomeric detergent concentrations can be realized
by making bicelles with lower-cmc detergents (e.g., C
7
-DHPC; [
198
]), although
the utility of this bicelle type for solution NMR applications has yet
a
IIb
b
to be
demonstrated.
