Chemistry Reference
In-Depth Information
cells,
[
24
]
use of Semliki Forest virus-infected cells
[
25
]
and expression in the yeast
Pichia
pastoris
.
[
26, 27
]
All of these systems are capable of yielding sufficient quantities of folded,
functional membrane proteins for ligand screening and structural studies. Unfortunately
none is perfectly general and the rate-limiting step remains finding the best system for a
particular target of interest.
6.3.2 The Membrane Environment
Membranes are structured as stable phospholipids bilayers which delimit the boundaries of
the organelle or the cell. The membrane provides an environment where chemical signals
can be emitted and detected, where energy can be converted into inter- and intracellular
functions and through which materials can be transported. For all these activities, there
are complex networks of interactions between the membrane-associated proteins, such
as receptors, ion channels and enzymes, and the ligands which stimulate or inactivate
them. The membrane itself plays more than a passive role in these processes. Current
understanding suggests that interaction between the membrane and embedded proteins is
at least required for and may regulate protein function. Therefore, the ultimate goal of
research in our group is to be able to perform NMR-based ligand screening studies on
membrane proteins in their native environment. However, in the light of the discussion
in the preceding section, it is clear that this is not yet possible and therefore membrane
proteins must be recombinantly expressed and purified. Given the intimate interaction
between protein and membrane, functional solubilization represents a major hurdle.
In order to retain functionality of a membrane protein, it is imperative to refold it or
reconstitute it into a synthetic lipid environment which mimics the properties of its natural
membrane as closely as possible.
[
28
]
Integral membrane proteins must be solubilized before
being purified and this often calls for addition of detergents after the initial centrifugation
steps. For example, the potassium channel KcsA was extracted from the cell membrane
by addition of Foscholine-12 prior to purification using IMAC and gel filtration chroma-
tography.
[
29
]
Transmembrane proteins have large hydrophobic domains which can cause
aggregation during purification. This can be avoided by using high concentrations of urea
to prevent random folding before reconstitution in lipids.
[
30
]
These solubilization and puri-
fication steps are important because the success of lipid reconstitution depends on the state
of the protein at this point. Organic solvents are the simplest approach to mimicking a mem-
branous environment, but it has only been possible to use them with proteins with stable
native folds such as ATP synthase
[
31
]
or colicin E1 immunity proteins.
[
32
]
The simplest true
mimic of amembrane occurs when ionic or nonionic surfactants in organic solvents or water
create micellar vesicles.
[
33
]
Micelles, which are 10-100 kDa in size when there is low ionic
concentration, are very convenient since they are readily formed and can be used to solubil-
ize membrane proteins in a monomeric form amenable to high-resolution structural studies.
To date, all TINS screening has been applied to micelle-solubilized membrane proteins.
However, due to, at least in part, the monolayer and the extreme curvature of micelles,
they are only rarely compatible with native functioning of membrane proteins. Surfact-
ants used for such preparations include, but are certainly not limited to, sodium dodecyl
sulfate (SDS), cetyltrimethylammonium chloride and bromide (CTAC and CTAB), lyso-
phosphatidylcholine (LPC), Triton X-100 and dodecylphosphocholine (DPC).
[
34
]
For NMR
studies, deuterated surfactants are at least convenient and many times may be required. At
