My research interests center around a few key questions:

How do DNA-binding proteins discriminate against non-cognate sites?

While the current database of protein-DNA complex structures determined by crystallographic and NMR methods has contributed greatly to our understanding of the positive forces involved in the
high affinity binding of DNA by sequence-specific DNA-binding proteins, our understanding of the mechanisms that these proteins use to discriminate against the myriad of similar but incorrect sites in the genome lags far behind. The central theme of this project is the application of crystallographic methods to elucidating the mechanisms involved in discrimination against sub-optimal ligands; i.e., What is the structural basis for the higher energy state of the complex between a DNA-binding protein and a minimally-suboptimal DNA oligonucleotide?


Can small molecules be found or designed to interfere with sequence-specific protein-DNA recognition?

The apparently fragile nature of the protein-DNA interface, coupled with the delicate balance between affinity and specificity, suggests that it may be possible to find or devise small molecules which could interfere with sequence-specific protein-DNA recognition. The central theme of this second project is to discover, design, and characterize these substances and determine their mechanisms of action, preferentially targetting the DNA-binding surface of the protein rather than the DNA.


Collaborative Projects:

uvsY - DNA Prof. Scott Morrical and Hans Beernink, Ph.D.

Proteins such as gp32 from phage T4 stabilize and protect single-stranded DNA by tightly binding to and coating it - so tightly that the subsequent binding of the replication and recombination machinery is hindered. Additional proteins are required to mediate the efficient loading of the R & R machinery onto gp32-coated ssDNA, such as the uvsY protein from phage T4. The current model for the action of uvsY is that it binds to the coated ssDNA, disrupts the cooperative binding of gp32 on DNA, and mediates the exchange of the filamentous recombination protein uvsX for the displaced gp32. Towards understanding the detailed molecular mechanism of this process, Hans Beernink crystallized the uvsY-ssDNA complex while he was a graduate student in Scott Morrical's lab. As a post-doc, Hans is continuing the crystallographic structure determination of this complex in a collaboration between the Morrical and Rould Labs.


Structural Studies of Myosin Function Prof. Kathy Trybus, Prof. Susan Lowey, and Terri Messier

The 3-dimensional crystal structure of a smooth muscle motor domain-essential light chain complex with several bound nucleotides has provided the first snapshot of the molecule in the pre-power stroke conformation (Cell 94: 559-571, 1998). These studies are being extended to other nucleotide analogs, as well as to mutants that have impaired function or which are stalled at various stages of the crossbridge cycle. A large body of supporting biochemical and mechanical data has been obtained for the proteins that have been chosen for the structural studies. Crystallization of double-headed heavy meromyosin is also being pursued, with the goal of understanding the head-head interactions that are required for phosphorylation-dependent regulation of this motor's activity. We are also pursuing the crystallization of an unconventional myosin motor (myosin V and its calmodulin-binding domain), which is involved in vesicular transport within the cell.