Biology Reference
In-Depth Information
II. Amphitropic Proteins
Amphitropic proteins
[10]
are a relatively new class whose importance is rapidly
becoming more apparent. Unfortunately there is a lot of overlap between the peripheral
proteins that are bound to membrane surface phospholipids, ecto and endo integral proteins
and amphitropic proteins. Amphitropic proteins can alternate between conformations that
are either water-soluble or weakly bound to the membrane surface through a combination
of electrostatic and hydrophobic forces. Since the membrane interaction is reversible, an
amphitropic protein co-exists as both a free globular protein and a membrane bound protein.
The water-soluble globular form is converted into the membrane-bound form following
a conformation change induced by phosphorylation, acylation or ligand binding that exposes
a previously inaccessible membrane binding site. Membrane interaction is often based on
production of a hydrophobic loop of amino acids, sometimes an
a
-helix, that can penetrate
into the lipid bilayer.
There are many important amphitropic proteins that are integrally involved in cell
signaling events. Examples include Src kinase, protein kinase C and phospholipase C.
Upon binding with the appropriate substrate, conformation of the globular form of the
enzyme is altered, exposing a hydrophobic segment on the protein that inserts into the
membrane where it comes into close association with its membrane target. For example, in
E. coli
globular puruvate oxidase binds to its substrate pyruvate and cofactor TPP (thiomine
pyrophosphate), exposing a hydrophobic helix in the protein. The modified enzyme then
binds to the membrane transferring electrons from the cytoplasm to the electrom transport
chain in the membrane. The amphitropic protein category also includes water-soluble
channel-forming polypeptide toxins including colicin A and
a
-hemolysin.
III. Integral Proteins
It is estimated that 20
30% of all cellular proteins are integral and these proteins are
tightly and permanently bound to the membrane
[11]
. While peripheral and amphitropic
proteins are weakly bound to the surface of membranes and can be readily dissociated
producing lipid-free globular proteins, integral proteins are integrated into the membrane
hydrophobic interior and can only be removed by more drastic means. Historically, integral
proteins have been separated from membranes through acetone and other non-polar organic
solvent extractions, chaotropic agents or detergents.
The earliest attempts to isolate integral proteins involved the brute force method of dis-
solving away the membrane lipids by use of cold acetone. Proteins precipitate into a white
powder called an acetone powder. Of course the proteins are completely denatured and
virtually worthless for biochemical analysis. Chaotropic agents have been successfully
employed to isolate some still functional integral proteins. Chaotropic agents function by dis-
rupting water structure, thus eliminating the hydrophobic effect that is the major force stabi-
lizing membranes. Although not as harmful as acetone, chaotropic agents may also denature
membrane integral proteins. Chaotropic agents are highly water-soluble solutes that essen-
tially pull all of the water away from a membrane, destabilizing it. The best example of a cha-
otropic agent is 6 to 8 M urea. Other examples include high concentrations of guanidinium
chloride, lithium perchlorate and thiocyanate. At present the best method for isolating
e
