Chemistry Reference
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for nanodisc-embedded membrane proteins have shown broad
1
H line widths [
153
,
204
,
207
]. Rapid axial rotation of smaller membrane proteins within the bilayer can
significantly reduce these line widths [
205
]. Unfortunately this is unlikely to occur
for the larger membrane proteins that are more prone to require reconstitution in a
detergent-free lipid environment. Consequently, the most common application for
nanodisc preparations in solution NMR to date has been to provide a reference 2D
correlation spectrum of a bilayer-reconstituted sample to allow comparisons with
results obtained in less bilayer-like solvents [
153
,
205
,
209
]. The GPCR CC-
chemokine receptor 5 (CCR5) has also been reconstituted in nanodiscs, allowing
its interaction with its chemokine ligand to be studied by solution NMR [
210
].
Nanodiscs have additionally proven useful for the study of interactions at mem-
brane surfaces (e.g., peripheral phosphoinositide-binding proteins [
206
]). Mean-
while further developments in nanodisc formulations and protein labeling may help
to expand the utility of these complexes for solution NMR in the future.
3.3.2 Amphipols
A distinct class of surfactant that is also being developed for a range of membrane
protein applications is the amphipathic polymer, otherwise known as the amphipol
[
211
]. These molecules are typically comprised of an amphipathic “backbone” with
hydrophobic branches interspersed with polar or charged groups (reviewed in
[
212
-
214
]). The result is a polymer that could be thought of as a unimolecular
micelle with covalent bonds linking polar headgroups (Fig.
3
). The equilibrium
between monomeric and micellar states that characterizes unlinked detergent
solutions is avoided by the covalent linkages, and only a few amphipol molecules
are required to envelop and solubilize the protein target.
Transfer of a membrane protein into amphipols can be achieved by sub-cmc
dilution of a detergent-solubilized sample into an amphipol solution. The increase
in entropy that results from the release of multiple detergent molecules upon
amphipol binding makes the exchange highly favorable [
215
], allowing functional
reconstitution of a wide range of membrane proteins [
211
,
216
-
218
]. Particularly
impressive is the ability of amphipols to refold SDS-solubilized GPCRs to greater
yields than could detergent/lipid mixtures that had been identified from extensive
screening experiments [
219
]. This allowed the structure of a GPCR ligand in its
receptor-bound state to be determined by solution NMR [
220
]. Meanwhile, feasi-
bility for solution NMR of the solubilized protein itself has also been demonstrated
with the transmembrane domain from OmpA [
221
]. While a small increase in
1
H
line widths was observed in the amphipol relative to the C
6
-DHPC spectrum, this
could be attributed to the slightly larger size of the amphipol-OmpA complex, along
with chemical exchange processes. These results with a first-generation amphipol
provide an encouraging indication of the potential that future amphipols could have
for membrane protein structural biology. The development of amphipols that can
remain soluble under more acidic conditions that decrease rates of amide proton
