Autumn Quarter 2006 - Microscopic Reversibility in EnHD

The first step in unfolding EnHD is helix III pulling away from helices I and II and exposing the hydrophobic core.

My first rotation as a graduate student was in the Daggett Lab, investigating whether or not protein folding obeys the principle of microscopic reversibility. In the context of protein folding, this means that under the same conditions the unfolding pathway is the exact reverse of the unfolding pathway. I used a molecular dynamics simulation around the T_m of the Engrailed Homeodomain (EnHD) to investigate whether the protein went through the same transition state in unfolding and refolding and whether the path was the same.

McCully ME, Beck DAC, Daggett V. Microscopic reversibility of protein folding in molecular dynamics simulations of the engrailed homeodomain. Biochemistry 47(27): 7079-89, 2008. [DOI]

Winter Quarter 2007 - Designing a Retroaldolase

The designed active site with the ligand in yellow and catalytic residues in orange.

My second rotation was in the Baker Lab, using Rosetta to computationally design an enzyme to carry out the retroaldolase reaction. I began with the ligand, shown in yellow, and the optimal relative positions of the three catalytic residues, in orange. Then, using Rosetta, I was able to place this structure into the pocket of several protein scaffolds. After rounds of optimizing the catalytic geometry and minimizing the energy of the structure, I reached my final theoretical design.

Spring Quarter 2007 - Predicting HIV Protease Inhibitor Binding Energies

HIV Protease with an inhibitor bound in the active site.

I spent my last rotation in the Samudrala Lab where I worked on testing and expanding a protocol developed in the lab for predicting binding energies of inhibitors for HIV Protease. HIV Protease is essential for HIV to infect its host and spread, so if we design an inhibitor that binds the protease with high affinity, we can potentially inhibit the virus. A lot of the work on inhibitor design is now done computationally since it is relatively inexpensive. However, this necessitates an accurate, high-throughput testing procedure to see how well the designed inhibitor will bind. My project was to continue work on optimization and testing of the protocol one of the postdocs previously developed.

Summer 2004 - PIP₂ and Actin-Regulating Proteins

Profilin (purple and yellow cartoon) with a truncated version of PIP₂ (red and blue sticks) docked in its binding location according to some preliminary results.

Within all eukaryotic (plants, animals, yeast, you) cells is a network of filaments responsible for maintaining the cell's shape as well as allowing the cell to move and replicate, among other things. These filaments are made up of individual actin monomers, which are proteins. Whether or not the actin is polymerizing (making the filaments longer by adding on more monomers) or depolymerizing (taking monomers off) is regulated by other proteins such as profilin, cofilin, and capping protein. These proteins are further regulated by phospoinositol-4,5-bisphosphate (or PIP₂ for short), which is a lipid in the cell membrane.

The goal of my research was to find where and how PIP₂ binds to the actin-regulating proteins using computer simulations and docking. Since proteins are flexible structures, the crystal structure is not the best representation of the many conformations the protein can take in vivo. Think of it as the difference between a picture and a movie. Taking the crystal structure of one of the proteins of interest (such as capping protein), I used the molecular dynamics software suite, GROMACS, to simulate the protein. I took several frames from this "movie," and used the program AutoDock to find the possible binding locations for PIP₂ on each of these proteins.

This research was supported by a fellowship from the Howard Hughes Medical Institute.

HHMI Summer REU Symposium Abstract

Kim K, McCully ME, Bhattacharya N, Butler B, Sept D, Cooper JA. Structure/function analysis of the interaction of phosphatidylinositol 4,5-bisphosphate with actin-capping protein: implications for how capping protein binds the actin filament. J Biol Chem 282(8): 5871-9, 2007. [DOI]

Summer 2005 - A22 and MreB

The binding location of A22 on MreB overlaps with the ATP-binding site, providing a structural explanation for why A22 inhibits microtubule formation.

In 2005 I was back in the Sept Lab working on a new docking project. The protein I was looking at was MreB, a protein similar to actin, found in prokaryotes (bacteria and other single-celled organisms with no organelles), and the ligand is A22. A22 was found to halt cell division by binding to MreB and inhibiting microtubule formation (they help separate the chromosomes into the two daughter cells). Again, my goal was to use computer simulations to find out where A22 binds as well as to suggest fluorescent analogs of A22 to use for experimental study of MreB.

Bean GJ, Flickinger ST, Westler W, McCully ME, Sept D, Weibel DB, Amann KJ. A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. Biochemistry 48(22): 4852-7, 2009. [DOI]