Research Description:

Actin-Binding Properties of CWIP

Last September in the Proceeding of the National Academy of Sciences, biologists at Harvard published a paper claiming they synthesized a peptide which they called CWIP, that could inhibit the protein N-WASP and thus stimulate the formation of actin filaments. This attracted our attention because although N-WASP is an important protein that regulates changes in actin, we doubted a peptide could cause indirect effects. I began to test a new hypothesis, that the CWIP affected actin polymerization directly, not indirectly through N-WASP as previously claimed.

Actin is an important protein in muscles as well as many unicellular organisms, which depend on it for motility, by the formation of actin filaments. We presume that CWIP, a 14 amino acid peptide with a dimeric structure, binds to actin in two places. Initial tests I preformed involved using pyrene-labeled actin added to the CWIP. Pyrene fluoresces with the presence of filamentous actin, thus more polymerization results in higher readings. It was immediately obvious that the CWIP showed a dose-response: the more CWIP added, the more actin polymerized. However, an error I made during a version of this experiment a few months ago proved invaluable. I forgot to add the labeled pyrene-actin and instead set up the experiment with unlabeled actin, but obtained the SAME results. This means for the past few months we were not detecting F-actin but instead, the effects of light scattering. This is a very unique characteristic of a peptide. Also, around this time, another interesting discovery was made. I decided to read my samples 24 hours after I made them (I usually read them immediately after they are made), and discovered that big changes take place with the actin overnight. The CWIP has an unusual effect in that it causes actin to aggregate in clumps by just sitting out, without being centrifuged. This planted seeds of doubt that CWIP behaves as initially hypothesized: instead it interacts very strongly (and uniquely) causing actin to just aggregate in disorganized clusters.

Since these discoveries, we have been redesigning the CWIP by changing some of its amino acids, to alter its interactions with actin. Once we successfully modify CWIP so that it doesn’t interact too strongly (and cause aggregation) with the actin, I plan to characterize it’s binding activity (where it binds, how it might change actin structure), as well as its true effects on actin polymerization.