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Dawson, with research associate Rema Balambika, and graduate
student John Blankenship also have several projects involving
modification of the amide backbone of proteins—something
he refers to as "backbone engineering."
Backbone engineering basically entails modifying the basic
structure of the amino acid, for instance, in order to remove
the ability of the backbone to form hydrogen bonds with other
parts of the protein molecule (this "H-bonding" is an important
factor that drives folding and stabilizes the three-dimensional
structures of proteins).
In general, this is an example of how protein synthesis
provides a useful tool for studying protein folding and the
formation of hydrogen bonding networks that are believed crucial
for it. By changing amino acid "amides" to "esters," Dawson
and his colleagues are able to knock out individual hydrogen
bonds, and they can then perform refolding studies to see
the effect of the removal of these bonds on the structures
of the proteins.
One specific question that they have asked is what role
such backbone H-bonds play in the formation of alpha helices.
"We think of the alpha helix in terms of its hydrogen bonding
network," says Dawson, adding that the synthesis of proteins
with or without these bonds allows them to go to the particular
bond in which they are interested and ask whether it is important
for folding.
Another variation of this, which they performed recently,
involves putting in esters to replace the amino backbone about
every three residues in stretches of proteins that form alpha
helices. Because of the nature of the alpha helix, this process
allows them to wipe out all the hydrogen bonds along a single
"stripe" of the helix.
They found that a protein with three ester bonds still folded
to form a functional protein. This result suggests that alpha
helices do not require the nucleation of multiple hydrogen
bonds in order to fold. Says Dawson, "This was a little surprising."
Tying Proteins in Knots
Looking at another question of basic protein biophysics,
another large area of research in Dawson's laboratory that
has benefited from this chemical control has been the development
of what are known as catenanes—circular, interlocking
protein rings.
These protein rings are basically short threads of about
40 amino acids that can be joined at the ends to make closed
circle loops. In principle, these loops can be joined together
to form a chain-linked protein polymer. They might turn out
to be useful self-assembling materials that could form a two-dimensional
sheet or a three-dimensional lattice. Furthermore, given the
level of chemical control that Dawson and his colleagues could
wield over these materials, it is possible to put metals or
non-peptide binding proteins on the molecules site-specifically.
"We think this could be a great way to assemble proteins
in a defined manner," says Dawson.
The first catenane that he and his laboratory designed was
unusually stable—so stable that the catenane was still
folded in boiling water and formed a self-associating "dimer"
of catenanes. This prevented further analysis. "It was easy
to make but hard to study," says Dawson.
So Dawson's graduate student John Blankenship, who is receiving
his Ph.D. from TSRI's Kellogg School of Science and Technology
next month, fixed the problem. Blankenship designed a catenane
based on a domain of the "tumor suppressor" protein p53. This
system formed a single, isolated catenane consisting of two
interlocking rings. This protein catenane was significantly
more resistant to unfolding or proteolysis than the original
p53 protein, and more so than any other protein cross-link
characterized to date.
Additionally, Blankenship discovered that the catenane can
be assembled in a step-wise fashion. "John found that if he
cyclized one of the pieces first, he could thread the other
piece through it and that the threading was very efficient,"
says Dawson. This enables the construction of heterocatenanes
and other, more complex structures, as the sequence "threaded"
through the cyclic protein need not be the same. Dawson adds
that they are currently trying to characterize the threading
process in more detail and determine how the process of threading
alters the protein folding pathway.
Understanding how the threading process effects protein
folding could provide a tool for manipulating the folding
of p53—or other intertwined proteins—in the cell.
In principle, one could synthesize cyclic peptides that would
interfere with some protein involved in cancer or another
disease state. "There are several [cases] where you could
actually inhibit a protein-protein interaction through threading,"
says Dawson.
And finally, the work demonstrates that making knotted proteins
is feasible. The fact that proteins fold into stable three-dimensional
conformations is well known, but what was less understood
a few years ago was if and how proteins could
thread themselves through a loop and make a knot. Most folded
proteins, if they were grabbed by the N-terminus with one
tiny molecular hand and the C-terminus with another and pulled,
would unravel into a single thread. Very few would result
in a knot.
In general, the success of this project was gratifying,
says Dawson. "It wasn't clear that [the peptides] would be
able to thread at all—it was surprising how well it worked."
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