Welcome to my webpage.
I am a research associate at the Perelman School of Medicine at the University of Pennsylvania, in the Department of Biochemistry and Biophysics. I work with Gregory Van Duyne, using structural and biophysical methods to study nucleoprotein assemblies that underlie basic biology and the molecular basis of disease.
I have been at Penn for the past 20 years, not only as an undergraduate and graduate student, but also postdoctoral fellow and now research scientist. I currently reside in South Jersey with my wife of 16 years and my two children (10 and 8), along with two dogs and two cats. My wife is an emergency veterinarian at Mount Laurel Animal Hospital.
In addition to my science cap, I also wear a musical one, serving as an assistant with the University of Pennsylvania Band. I am an avid music arranger and Penn basketball fan. In addition to the undergraduate mission, I spearhead the group’s community and high school outreach efforts, including the summer band camp and high school honor bands.
A central theme of my current research is lateral DNA transfer: the study of the structure and mechanisms of enzymes that catalyze site-specific recombination and retroviral integration. Throughout my career, I have integrated an array of biochemical, biophysical, and structural methods to better understand the molecular properties of biological macromolecules related to these processes. My primary training is in x-ray crystallography; during my time in the Van Duyne laboratory, I have complemented this skill set with additional training in biochemical and biophysical approaches important to the study protein-protein and protein-DNA interactions. These techniques include gel-based assays for binding and activity, analytical centrifugation (AUC), multi-angle light scattering (MALS), small-angle x-ray scattering (SAXS), small-angle neutron scattering (SANS), and x-ray footprinting (XF). I have applied this repertoire of techniques to the study of large nucleoprotein assemblies that regulate the lateral transfer of genetic information, including the retroviral intasome (HIV, prototype foamy virus), site-specific recombination intermediates (Cre, TP901-1, large serine recombinases), group II introns, and nucleosomes.
Small-angle scattering has re-emerged over the past decade as an important technique for the study of macromolecules, due in large part to its ability to reliably reconstruct low-resolution solution structure. I have developed a very strong interest in the application of this method to the study of protein-DNA complexes and large macromolecular assemblies. Combined with other techniques, this is a powerful method which serves to understand solution structure and the hydrodynamic properties of larger macromolecular assemblies, especially where atomic structures of the component parts are known. .
The AIDS pandemic has infected and killed tens of millions of people worldwide over the past 25 years. Hence, the design of effective pharmacological treatments remains of paramount importance. HIV’s genome encodes three enzymes: reverse transcriptase (RT), protease (PR), and integrase (IN). Because integration of viral cDNA into the host genome is critical to the retroviral life cycle and since there are no known cellular enzymes that closely resemble IN in sequence or function, inhibitors of IN have the potential to be relatively nontoxic and very effective.
One focal point of my work has been the enzyme Cre recombinase, a site-specific recombinase from bacteriophage P1. This 38kD protein specifically recognizes and catalyzes strand exchange between identical 35 base pair sequences called loxP. These sites are composed of two recombinase-binding elements (RBEs) arranged as inverted repeats, surrounding a central strand exchange (crossover) region. The phosphoryl transfer strand exchange reaction occurs within the central 6 base pairs of the crossover region; the asymmetry of this region provides directionality to the site. In an event called synapsis, two loxP sites are brought together by four Cre monomers to catalyze site-specific recombination in a sequential process. Shown above is a crystallographic snapshot of the Cre-loxP synaptic complex. Over the past few decades, Cre has become a powerful tool for genomic manipulation, and remains the paradigm for study of the site-specific tyrosine recombinases. Key to the development of novel recombination tools for applications in biology is a detailed structural and biochemical understanding of the reaction catalyzed by these enzymes. The challenge in studying the biochemistry of the Cre system lies in the reversibility of the complex reaction catalyzed: every step in the recombination pathway is reversible, and the enzyme passes through several distinct states before reaching completion. I have been examining catalytic mutants of the enzyme to dissect the mechanism of the phosphoryl transfer reaction. Wild-type enzyme and several active site mutants have been studied by biochemical and pharmacological methods in vitro and via x-ray crystallography. This investigation has allowed us to delineate several key interactions involved in Cre-mediated catalysis and to distinguish this enzyme from other members of the Type IB topoisomerase family.
Prostaglandin H2 Synthase-1
The topic of my thesis work was the integral membrane protein Prostaglandin H2 Synthase-1 (PGHS), a key drug target involved in the formation of eicosanoids. Using x-ray crystallography as a primary tool, my research focused on two facets of the structure and function of this enzyme: the structural basis of time-dependent inhibition of this enzyme by NSAIDs, and the structural interrelationship of the two active sites located within the enzyme. With Dr. Loll’s guidance, I determined six structures of Prostaglandin H2 Synthase-1 in complex with different ligands and alternative cofactors using synchrotron radiation available at NSLS Beamlines X8C and X25. Use of the enhanced x-ray flux at X25 in combination with improved cryofreezing conditions allowed for the determination of the first high-resolution structures of this clinically important drug target. (2 Å resolution).
Related Projects I've worked on:
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