Biology Reference
In-Depth Information
often using the same detergent used for protein isolation. If these two populations of deter-
gent micelles are mixed and the detergent removed by dialysis, gel filtration or preferably by
binding to non-polar polystyrene beads (Bio-Beads), proteoliposomes can be directly
produced [50,51] . In addition to this method, a variety of other methods rely on adding
the protein/detergent micelles to pre-made LUVs. These methods depend on first partially
destabilizing the LUV bilayers by either detergent or brief sonication before adding the
protein/detergent micelles. Partial bilayer disruption is necessary to prevent protein dena-
turation at the surface of tightly packed lipid bilayers. Several detergents have been used
as destabilizing agents, primarily low levels of Triton X-100. Tip sonication must be very brief
to prevent protein denaturation or may be replaced by gentle bath sonication.
Ideally, the proteoliposomes should be nearly detergent-free LUVs that contain the active
integral protein spanning the membrane lipid bilayer. However, one potential problem
concerns determining the protein's orientation in the membrane. Four orientations of the
newly reconstituted protein are possible. Suppose in the biological membrane the trans-
membrane protein exists with an A-end exposed to the aqueous cell interior and a B-end
exposed to the aqueous cell exterior. The possible orientations for the reconstituted proteoli-
posome are:
1. A-end inside, B-end outside (correct orientation).
2. B-end inside, A-end outside (reverse orientation).
3. A-end inside, B-end inside (bent orientation).
4. A-end outside, B-end outside (bent orientation).
During the process of reconstitution some orientations are more favorable than others. For
example, if the protein is rigid like those with multiple span helices, the protein will not be
flexible enough to exhibit the two bent conformations (3 and 4). Many plasma membrane
proteins have an extensive sugar component that in a living cell will always face the external
aqueous environment (the B-end in the example). If the sugar component is sufficiently large,
steric hindrance in the small LUV interior will favor proteoliposomes with the sugars on the
external surface (1 and 4). A sugar attachment containing sialic acid presents an additional
problem
high negative charge density. Multiple negative charges require more spacing
between the sugar components, favoring an external orientation. It is also possible to isolate
and reconstitute a protein with a very large antibody attached at one end. This end will exclu-
sively favor external orientations. Of course, once reconstituted, the antibody can be
removed, thus restoring function.
There are now countless numbers of integral membrane proteins that have been succes-
sively reconstituted into proteoliposomes. Single protein proteoliposomes allow for close
inspection of any lipid specificity and molecular mode of action that may be difficult to deter-
mine in complex, multi-component biological membranes. A few examples follow.
e
Ca ATPase
Ca 2 þ ATPase is one of the most studied of all integral membrane proteins [52] . This trans-
port protein (pump) was first discovered in 1966 and successfully reconstituted into proteo-
liposomes by the early 1970s [53] . The pump comes in two basic types, the plasma membrane
Ca 2 þ ATPase (PMCA) and the sarcoplasmic reticulum Ca 2 þ ATPase (SERCA). Both pumps
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