I am a research assistant professor at the Perelman School of Medicine at the University of Pennsylvania, in the Department of Biochemistry and Biophysics. I use structural and biophysical methods like X-ray crystallography, small angle scattering, analytical ultracentrifugation, and light scattering to study large macromolecular assemblies that underlie basic biology and the molecular basis of disease. I currently direct the Johnson Foundation Structural Biology and Biophysics Core Facility here at Penn.
I have been at Penn for the past 26 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 21 years and my two children (16 and 13), along with three dogs. My wife is an emergency veterinarian at Mount Laurel Animal Hospital.
In addition to my science cap, I also wear a musical one as part of the directorship of 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 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.
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.
Retroviral integrase (IN) catalyzes the incorporation of viral cDNA into the host genome. The design of effective pharmacological treatments remains of paramount importance to the treatment of HIV/AIDS, and detailed structural models of intact IN oligomers in their various states are essential to new structure-based drug design efforts. My work on the retroviral integrase (IN) has focused on the understanding of higher-order structure and oligomeric forms of the full-length integrase when bound to host factors and DNA, with the overall goal of determining the molecular details of the larger macromolecular assemblies that underlie the steps of retroviral integration and other stages of the viral life cycle.
My research has married X-ray crystallography and rigorous biophysical methods to approach these fundamental questions. These approaches have included the application of small angle X-ray and neutron scattering (SAXS/SANS), analytical ultracentrifugation, multi-angle light scattering, and molecular modeling. These studies have yielded understanding of the quaternary structure and stoichiometry of IN, IN-DNA, and IN-host factor assemblies.
Most recently these approaches have been brought to bear on an exciting new class of allosteric inhibitors (“ALLINIs”) that is able to inhibit IN via selective modulation of its oligomeric properties. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. In 2016, we reported a breakthrough in the structural biology of HIV Integrase: the first crystal structure of HIV-1 Integrase in complex with the ALLINI GSK 1264. To our knowledge, this is the first time full-length HIV-1 integrase has been crystallized. The structure shows GSK1264 bound to the dimer interface of the catalytic domain, and also positioned at this interface is a C-terminal domain (CTD) from an adjacent IN dimer. In the crystal lattice, IN forms an open polymer mediated by this interaction. Further studies of a panel of ALLINIs show that HIV escape mutants with reduced sensitivity commonly alter amino acids at or near the inhibitor-mediated interface, and that HIV escape mutations often encode substitutions that reduce multimerization.
Small-angle scattering has re-emerged over the past two decades 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. .
Related Projects I've worked on:
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