Joseph Mcnulty Email & Phone Number

Los Angeles County, California, United States

My post-doctoral research was deeply involved with engineering DNA binding behavior and specificities for zinc finger domains. Early on, I developed a custom bacterial two-hybrid system to build and test novel zinc finger chimeras to eukaryotic interaction domains in order to create 'switchable' DNA binding domains that could be activated with a second exogenous factor. My more recent work (which we are currently preparing to publish in spring 2011) centers on engineering novel specificity for zinc finger domains using a bacterial one-hybrid system. We developed a new approach for building new zinc finger subunits using focused recombinant DNA libraries coding for zinc finger 'recognition helices' that encompassed the desired target binding specificities. Our innovation was to combine this approach with a novel architecture that has proven successful in targeting DNA sequences previously resistant to traditional selection methods. Zinc finger nucleases created using these domains are among the most effective and least toxic in our existing repertory, and we characterize their 'true' in vivo activity by considering their activity at several potential off-target sites. Other projects of note include using extensive recombinant libraries (~10^8 members) to probe the binding preferences of a previously undescribed zinc finger scaffold belonging to a 'rare' class that bound adenine-rich sequences with high specificity, and a concerted effort towards generating targeted germ-line "knock-in" insertional mutations in zebrafish using zinc finger nuclease - stimulated homologous recombination.

This opening phase of my post-doctoral work immersed me into the worlds of synthetic and systems biology, and marked my entry into molecular biology techniques. The first project attempted to integrate modular allosteric autoinhibition to a beta-sheet rich protein by destabilizing key contacts at the terminii of the domain sequence. Subsequent efforts were directed towards initiating the zinc-finger engineering work that formed the bulk of my post-doctoral research.

I completed the NMR structure determination of the "ASIP-YY" variant that had been engineered via the double-tyrosine mutation to exhibit the folding behavior of the highly homologous AGRP domain (see below for abbreviation descriptions). The high disulfide content (over 20% of the residues are cysteines, all involved in 5 disulfide bonds) present in both domains precluded relying on a straightforward homology-modeling approach, as multiple 3D-cystine knot arrangements were easily possible. Ultimately, discovery of a cis-trans proline isomerization in a proline-rich loop allowed for the determination of two separate families of NMR structures, both of which were consistent with the overall fold of the homologous AGRP protein. This helped provide a foundation to consider both protein domains in terms of an 'active' surface on a supporting 'scaffold' with a highly similar fold.

My Ph.D. thesis research in Glenn Millhauser's lab at UC Santa Cruz involved the spectroscopic analysis of chemically synthesized peptides and protein domains, broken into two distinct and fully developed projects. My early work revolved around the spectroscopic analysis and synthesis of spin-labeled aqueous model helical peptides, while my second body of work focused on the solid-phase chemical synthesis and NMR structure determination of the C-terminal domain of the Agouti signaling protein (ASIP). Successful synthesis and folding of this domain required a double mutation, incorporating two tyrosines from the homologous Agouti-related protein to help stabilize and direct the oxidative folding of the synthetic peptide to the proper 3-dimensional arrangement of cystine-disulfide bridges. The resulting "ASIP-YY" molecule was shown to be biologically active, amenable to NMR structure determination, and has served as a reagent and starting point for a wide variety of studies through to the present day.

Ph. D., Chemistry

Bs, Chemistry, Biology, Mathematics

Activities and Societies: Alpha Chi Omicron Honor Society

Bs, Chemistry & Biology (Double Major)