(page 2 of 2)

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."


1 | 2 |



Design of an interlocked protein.
Click to Enlarge