Chemistry Reference
In-Depth Information
3.3 New Developments in Membrane-Mimetic Solvents
for Solution NMR
While micelles and bicelles have served as the membrane-mimetic solvent for the
majority of solution NMR studies on integral membrane proteins, the identification
of suitable NMR-compatible solvents remains a formidable challenge. With
30
unique multi-spanning membrane protein structures determined by solution NMR,
there is a clear need to expand the current library of solubilization agents for this
purpose. In response to this problem, creative approaches are currently being
developed that may open the door to solution NMR for a wider range of membrane
proteins in the future. Some of the systems that have started to yield promising
results include nanodiscs, amphipols, and reverse micelles.
<
3.3.1 Nanodiscs
Nanodiscs are comprised of a bilayer containing ~130-160 lipids, maintained in a
discrete, water-soluble state by the association of two copies of the membrane
scaffold protein (MSP) from apolipoprotein A-I wrapped around the hydrophobic
rim of the bilayer [
199
-
202
] (Fig.
3
). Originally developed for the solubilization of
functionally active integral membrane proteins, they have since been used for solid-
state [
203
], and more recently, solution NMR applications [
153
,
204
-
207
].
Incorporation of an integral membrane protein in a nanodisc is usually achieved
by incubation of a detergent-solubilized sample with detergent-solubilized lipids,
along with MSP. Detergent removal, often with adsorbent beads, can promote the
spontaneous formation of nanodisc-reconstituted samples that remain stable for
many months [
202
,
208
]. However, a number of variables must be optimized when
formulating a reconstitution procedure, with the choice of detergent for membrane
protein solubilization being identified as a particularly critical one [
200
]. Even
when solubilized in detergents, a membrane protein may undergo irreversible
aggregation that cannot be rescued by reincorporation into a native lipid bilayer.
Consequently, high yield reconstitution requires a detergent that can minimize the
formation of these aggregates, often with the use of excess nanodisc-forming
components.
Once reconstituted into a nanodisc, the membrane protein is embedded in a
bilayer phase that shares some similarity to that in the bicelle. However, the absence
of detergent in the final nanodisc preparation can prevent the folding/stability
problems that may be introduced by the significant concentrations of monomeric
detergent in bicelle and micelle solutions. The nanodiscs themselves are also more
stable than bicelles, and can be subjected to the same manipulations as soluble
proteins (e.g., lyophilization, chromatography, concentration, etc.) [
200
]. However,
there is a significant trade-off in that nanodisc sizes tend to be on the large side for
NMR applications, at ~200 kDa. While this remains within the accessible range for
relaxation-optimized NMR experiments (see Sect.
4.2
), most solution NMR spectra
