Chemistry Reference
In-Depth Information
exchange with solvent, along with methods that improve the homogeneity of
prepared samples will be particularly important for this application.
3.3.3 Reverse Micelles
A fundamental restriction for solution NMR of solubilized membrane proteins is
imposed by the large size of the complexes they make with detergents and lipids,
since they usually exhibit slow molecular reorientation rates, giving rise to signifi-
cant line broadening and decreased coherence transfer efficiencies. To reduce the
influence of molecular mass on rotational correlation times, and hence NMR
spectral quality, the Wand group has pioneered the use of reverse micelles to
encapsulate water-soluble proteins [
222
-
224
]. When dissolved in an organic sol-
vent of low viscosity, molecular tumbling rates of these complexes are significantly
enhanced compared to those in water. When the increase in molecular reorientation
rates exceeds the decrease that comes from the added mass of the reverse micelle,
significant improvements in size-sensitive NMR spectral properties can result
[
225
]. For a reverse micelle in pentane this benefit may not appear for a water-
soluble protein until its size exceeds ~50 kDa; in contrast, membrane proteins
already require detergents for solubilization. Moreover, the benefits of reverse
micelle encapsulation can be dramatically improved by the use of solvents with
lower viscosities such as butane and propane, which require only slightly elevated
pressures to maintain the liquid state in regular NMR tubes [
222
]. Even larger
advantages can be realized by the use of liquid ethane under elevated pressures
(~4000 psi) prepared through the use of a special apparatus [
226
]. In this solvent a
>
100-kDa protein can be conferred with molecular tumbling properties rivaling
those of a 10-kDa protein in aqueous solution.
Although reverse micelle systems were first developed for the study of water-
soluble proteins, their application to integral membrane proteins has since been
demonstrated with gramicidin A (gA) in a dioctyl sulfosuccinate/pentane system
[
227
]. The pattern of NOEs obtained for the gA peptide dimer in this medium was
consistent with its native
b
6.3
-helix. In addition, the intermolecular NOEs showed
preservation of native N- to N-terminal intersubunit hydrogen-bonding interactions
within the hydrophobic phase. Introduction of gA into this medium was relatively
straightforward, since this peptide can be isolated in a lipid and/or detergent-free
form and then directly introduced into the reverse micelle system. This was a
critical advantage, since the standard reverse micelle systems formulated for
water-soluble samples are not compatible with the detergents normally used to
purify larger membrane proteins [
225
].
To extend the utility of reverse micelles to those samples that require detergent
or lipid to prevent aggregation and promote folding, reverse micelle formulations
have recently been introduced that use detergents capable of forming regular
aqueous-phase micelles (e.g., LDAO, dodecyl- or cetyl-trimethylammonium bro-
mide (DTAB or CTAB, respectively)) [
228
]. These can be used to solubilize
membrane proteins in the aqueous micelle conditions usually used during purification.
