Biomedical Engineering Reference
In-Depth Information
transmembrane domains of three E. coli histidine kinases, including a four-
helical bundle.
98
12.2.6 Directed Evolution
Whilst there are many different expression systems available for membrane
protein production, the inherent instability of membrane proteins, particularly
the GPCR class, remains a significant barrier to their high-level expression and
biophysical characterisation. For crystallisation of GPCRs, techniques such as
fusion to T4 lysozyme
99
or the Fab antibody fragment
70
were required. Whilst
such methods may be successful for X-ray crystallography, the significantly
increased size of these complexes would compromise NMR studies. A more
appropriate method for NMR is likely to involve directed evolution of
receptors for greater stability, allowing expression of more receptors per host
cell and also greater stability in vitro. This has been demonstrated for the b
1
-
adrenergic receptor, which was thermostabilised in the antagonist-bound
conformation via six point mutations
66
enabling detergent solubilisation and
crystallisation. High-throughput methods including the use of fluorescent-
activated cell sorting
100,101
and streptavidin-coated paramagnetic beads have
been proposed.
100
Approaches such as this will support NMR studies of larger
membrane proteins, for which high-level expression and long-term stability
will be important factors to enable sufficient data collection for structure
determination.
12.3
Membrane Mimics
12.3.1 Detergents
Crucial to studying membrane proteins in vitro is finding an appropriate
solubilisation medium: this must maintain the membrane protein in its native
form, whilst typically having less complexity than the cell membrane, and also
providing long-term stability of the system (for a recent review see
Warschawski et al.
26
). For NMR, the chosen medium must have a relatively
small size in order to allow sufficiently fast tumbling and hence ensure sharp
lines and high-quality spectra. Typically, detergent micelles have been
favoured. (For structures of commonly used detergents see Figure 12.1.) The
micelle forms by orientation of hydrophilic detergent headgroups into the
polar solvent, whilst the hydrophobic hydrocarbon tails orient into the centre
of the micelle. Although micellar assemblies exist in various shapes,
102
for
simplicity these are often assumed to be close to spherical.
Detergent solutions are characterised by a monomer-micelle equilibrium
which forms above the critical micelle concentration, or CMC. Below this
concentration, only free monomers are found. Values can be determined for
the CMC and aggregation number of the micelle,
103,104
although these may be
affected by the conditions. Typically a protein is solubilised at detergent
