Biomedical Engineering Reference
In-Depth Information
improves interactions with hydrophobic regions, reducing the likelihoodof
protein aggregation.
119
A number of different headgroup structures have been investigated,
however, many have suffered from chemical heterogeneity. Recently,
surfactants with one, two or three glucose moieties have been presented,
overcoming the difficulty of heterogeneous preparations (Figure 12.1).
Increasing the number of headgroups results in an increasingly conical shape
of the (H)Fs leading to increasingly small micelles; surfactants with only one
headgroup were found to form large, cylindrical, polydisperse micelles.
Smaller homogenous aggregates were seen for those with two headgroups.
Similar properties were seen with three headgroups, however, these
detergents were found to be more destabilising, likely due to the high radius
of curvature.
120
Although such surfactants have only been recently described,
some studies are available. Cytochrome b
6
f is found to be dimeric in
surfactants with a single lactose or two or three glucose headgroups, similar
to low concentrations of dodecylmaltoside; however, whereas increasing the
DDM concentration causes a shift towards monomers, increasing (H)Fs
concentration maintains the dimer, demonstrating their milder proper-
ties.
120,121
Good stability (greater than or similar to detergents) is also seen
for bacteriorhodopsin (BR) and cytochrome b
6
f in F
6
-Monoglu or (H
2
)F
6
-
Diglu,
120,122
(see Figure 12.1 for structures) and protein refolding has also
been attempted.
23
The mild properties of (H)Fs may be of particular use in
cell-free synthesis; the mechanosensitive, pentameric ion channel, MscL, can
be synthesised in the presence of (H)Fs, which allow complete solubilisation
of the protein, without interfering with the translation system.
120,123
Although their use in NMR studies remains to be demonstrated, the mild
properties of (H)Fs may be of particular benefit for less stable systems and
also in cell-free expression.
12.3.3 Amphipols
Amphipols are amphipathic polymers designed, like the fluorinated surfac-
tants, to be less denaturing than many detergents, and to associate with
membrane proteins when only traces of amphipol are present in solution. This
approach aims to overcome the major limitation of studies using detergents
where, at the high protein concentrations required for structural studies,
especially NMR, high concentrations of detergents are needed, typically 50-
600 mM,
124
which can be inactivating. As a result of their mild properties,
amphipols cannot be used to extract protein directly from membranes.
Currently the most used amphipol is A8-35 (Figure 12.1), based on a short
polyacrylate chain, in which the carboxylates have been grafted with
octylamine and isopropylene chains, forming the hydrophobic regions of the
amphipol, whilst the free carboxylates interact with the water. Although the
solubility of this structure is reduced below pH 7, which may be limiting for
NMR studies,
125
15
N-labelled tOmpA were
TROSY spectra of perdeuterated
