Biomedical Engineering Reference
In-Depth Information
monomer.
The
smaller
size
eventually
enabled
successful
3D
structure
determination.
19
Subsequent work in our laboratory has investigated a number of other
detergents. pSRII was exchanged into decanoyl-N-hydroxyethylglucamide
(HEGA-10), n-nonyl-b-
D
-glucopyranoside (NG), as well as c6-DHPC (dihex-
anoylphosphatidylcholine), c8, c9 and c10-diPC (diphosphatidylcholine).
Difficulties with solubility precluded further work on c9 and c10-diPC, whilst,
as mentioned earlier, the high aggregation number for c8-diPC resulted in
extremely large micelles, rendering the spectra unsuitable. High-quality spectra
were achieved for c6-DHPC however, the sample lifetime was limited by faster
protein denaturation. For NG, properties similar to DDM were observed
[Figure 12.3(d) and (g)], suggesting a large aggregate size and possible presence
of a dimer. In contrast, HEGA-10 resulted in high-quality spectra, also
reflected in the fast translational diffusion rate, enabling assignment at 35 uC
[Figure 12.3(c) and (g)]. A potential trend suggests a characteristic of the
glucoside class of detergents is unsuitable for NMR studies of pSRII and
possibly other a-helical proteins. The uncharged headgroup and longer tail
may combine to result in micelles with reduced curvature and hence larger
aggregate size, and the milder properties of these detergents and large micelles
prevent the detergent from breaking larger aggregates. This may be a more
severe problem with other GPCRs which may form higher order oligomers,
putting them well out of reach of solution NMR. In contrast, c6 and c7-DHPC
have a charged headgroup and shorter chain tails giving a 'wedge-shaped
profile';
111
this is expected to result in smaller micelles, and the ability to break
oligomers. A similar profile is also observed for HEGA-10, which although
having a longer tail, has a larger headgroup, maintaining this wedge-shaped
profile.
12.3.2
Fluorinated Surfactants
Aside from the more classical detergents with hydrocarbon chains, a new
class of fluorinated surfactants with favourable properties have also become
available over recent years. Fluorinated surfactants are milder than their
hydrogenated analogues and do not have cytolytic properties. They are
unable to extract proteins from membranes as they do not partition well into
lipid membranes.
23,117
Since the bulky fluorinated chains, which are more
rigid than alkyl chains, interact less well with the methyl-covered
hydrophobic surface of proteins, they are less likely to destabilise protein-
protein interactions and their lyophobic character makes them less
delipidating than other detergents, preventing disruption of crucial protein-
lipid or protein-hydrophobic cofactor interactions.
23,118
Unfortunately such
mild detergents are likely to be unable to prevent protein aggregation as seen
in the case of the cytochrome b
6
f complex.
117
However, addition of a
hydrophobic tip e.g., a methyl or ethyl group to create hemifluorinated
surfactants (HFs) preserves the lyophobic character of these detergents, but
