Biochemistry
9:00 - 9:20 am
Determination of the Atomic Resolution, 3-D Structure of the Geosmin Synthase C-Terminal Domain
Ethan Pham
Institution: Mount St. Mary's University
Faculty Mentor: Patrick Lombardi
Terpenes are organic compounds comprised of isoprene units and are associated as metabolic waste products. The molecules' function are not entirely known. However, novel investigation suggests that terpenoids play a larger role in natural life mechanisms. There is interest in utilizing synthetic terpenes to innovate medicine and science. Synthetic terpene formation poses complexities due to their intricate formation mechanism, as such, there is interest on utilizing terpene cyclases; which form terpenes. Geosmin synthase, a terpene cyclase composed of two domains: a N and a C terminal domain, converts farnesyl diphosphate to geosmin, a terpenoid odorant that exhibits an earthy smell. Previous work has determined the N-terminal structure and inferences on function. The C-terminal’s structure has not been established and is crucial for complete geosmin synthase mechanism understanding. C-Terminal structural determination can infer its function within the reaction cascade. Elucidation of the cyclase's mechanism gives way for possible terpenoid synthesis advancement.
9:20 - 9:40 am
The Connexin 43 Carboxyl-Terminus Tail as a Src Inhibitor in Cancer Cells
Alec Buttner
Institution: Moravian College
Faculty Mentor: Anastasia Thevenin
Connexin 43 (Cx43) is a transmembrane protein that comprises Gap Junctions (GJs) - protein channels that connect neighboring cells, allowing for the transport of molecules and ions. The Cx43 carboxyl-terminal tail (Cx43CT) serves a tumor-suppressive function due to its interaction with Src, a proto-oncogenic signaling kinase. When Src interacts with Cx43CT in the Src binding region, two proteins (Csk and PTEN) are recruited to the Cx43CT to inactivate Src. Serine residues 279 and 282 are in the Src binding region of Cx43CT and are known to be phosphorylated by a number of kinases. We aim to define the optimal conditions for Src/Cx43 interaction and are testing how S279 and S282 phosphorylation affects this interaction. We utilize phosphomimetic Cx43 mutants at these sites, both in vitro and in cancer cells, in a series of co-immunoprecipitation and western blotting analyses. The results of our work will help inform our design of Cx43-based peptides for targeted delivery to cancer cells.
9:40 - 10:00 am
With or Without Oxygen: Amplification and Purification of the Periplasmic Nitrate Reductase Operon in E. coli
Yasmine Sakinejad
Institution: Salisbury University
Faculty Mentor: Katherine Miller
Periplasmic nitrate reductase reduces nitrate to nitrite in both anaerobic and aerobic conditions of microbial organisms. Analyzing the nitrate reductase gene’s catalytic subunits showed problems in previous lab experiments so improving experimental was needed. PCR was optimized using designed primers for the XL-1Blue strain of E. coli DNA and gel electrophoresis ensured primer binding and DNA amplification. The amplified DNA was purified, quantified and cloned, then competent XL1-Blue E. coli cells were transformed using the plasmids. Primer pairings of ECnapA733F and ECnapG197R, ECnapA1182F and ECnapB144R as well as ECnapF502F and ECnapH747R showed successful amplification with band sizes at around 312, 600 and 300 base pairs, respectively. Using the sequences obtained, ligation and transformation of E. coli exhibited plate growth for ligations with ECnapA1182F/ECnaB144R and ECnapF502F/ECnapH747R. Primer pairings ECnapA733F/ECnapG197 failed during ligation of plasmid despite high DNA concentration from PCR while ligation was successful for the other sequences. The next step is to send extracted sequences out for Sanger sequencing and continued exploration of other microbial organisms found in soil.
10:00 - 10:20 am
Purification of Corticosterone Binding Globulin for Quantification and Structural Analysis
Lauryn Ridley
Institution: Lehigh University
Faculty Mentor: Elizabeth Young
Photodynamic therapy (PDT) is a form of cancer treatment that uses light to kill cancer. In PDT, light irradiation of a photosensitizer (PS) localized in a tumor site leads to the production of singlet oxygen (also known as reactive oxygen species, ROS) within cells. ROS can trigger cancer cell death. The goal of this project is to evaluate several porphyrinoid-based PSs that are more potent than current PSs. Using transient absorption spectroscopy (TAS), the lifetime and triplet quantum yield of the PSs are measured determined to determine if these properties are correlated to the efficiency of the PS with respect to singlet oxygen production. Overall, we aim to determine the triplet lifetimes and quantum yields of the PSs and ultimately correlate that with the efficacy of pHLIP-PS conjugates in triggering cancer cell death.
10:20 - 10:40 am
Developing a reliable computational method to determine the allosteric sites to be targeted in finding non-aggregating allosteric inhibitors of PTP1B
Sarah Codd
Institution: Messiah University
Faculty Mentor: Anne Reeve
Type 2 diabetes is a serious metabolic disease with a growing need for improved therapeutics. Traditional antidiabetic drugs can assist with the maintenance of blood glucose levels in diabetics but are not optimal treatments due to side effects. PTP1B is a validated therapeutic target for type 2 diabetes, as it acts as a negative regulator in the insulin signaling pathway. Inhibiting PTP1B may allow for increased insulin sensitivity, leading to glucose homeostasis and improved metabolism. In the past 3 allosteric sites were identified as targets for PTP1B inhibition. This study analyzes the validity of these sites and the possibility of other allosteric sites through computational methods, organic synthesis, and enzymatic assays. A new allosteric site is identified and used to improve a computational method for testing possible inhibitors of PTP1B.
10:40 - 11:00 am
DNA Binding Interactions of Novel Dirhodium Compounds
Jessica Mickno
Institution: Moravian College
Faculty Mentor: Shari Dunham
Rhodium compounds are transition metal complexes largely underexplored for their biological activity. This work explores the anti-cancer potential of rhodium compounds by determining if they bind to DNA. DNA-binding can interfere with normal DNA processing and lead to cell death. In this study, dirhodium complexes with a variety of bridging ligands are incubated with dsDNA. Portions of each metal-DNA reaction are removed and filtered at certain time points to separate any unbound metal complexes from the larger DNA. The retained DNA is analyzed for concentration of metal complex bound using GFAAS. The ratio of metal to DNA is calculated. The amount of metal bound over time is plotted and used to determine the reaction rate, which is compared as the bridging ligands are changed. The results of these metal-DNA interactions will be presented and discussed with their relevance for the development of new potential anti-cancer drugs.
11:00 - 11:20 am
Fluorescent monitoring of divalent magnesium interactions in biochemical systems
Samantha Schauberger
Institution: Salisbury University
Faculty Mentor: Joshua Sokoloski
Magnesium ion interactions are ubiquitous in cellular systems and crucial to many cellular processes. Many of these interactions are often transient as in the case of the ion atmosphere around nucleic acids, making them difficult to measure and track. Due to this property, the use of a magnesium sensitive fluorophores, HQS, was our chosen method for tracking real time changes in [Mg2+]. Mg2+ binding to dsDNA, ATP, common buffer components and E. coli cells, was assessed by comparing the HQs fluorescence signals during MgCl2 titrations in the presence and absence of a cellular binding system. Affinities and binding stoichiometries can be estimated from these titrations. The ATP and DNA model systems exhibited strong Mg2+ binding interactions, while the bacterial interactions were more condition dependent. These results indicate the potential of the HQS assay for real-time observation of cellular magnesium metabolism.
11:20 - 11:40 am
The strategic unveiling and characterization of promising bioactive proteins
Christina McBride
Institution: Haverford College
Faculty Mentors: Lou Charkoudian and Eric Miller
Nature has the unparalleled ability to manufacture molecules of immense chemical diversity. Aptly named, these ‘natural products’ boast a wide variety of environmental, pharmaceutical, and industrial applications. Despite their availability, gaining access to these promising natural products is often difficult. Establishing effective methods for their identification, expression, isolation, and characterization provides an entryway to their bioactivity. This work focuses on two classes of natural products: polyketides (secondary metabolites produced by enzyme assemblies) and bacteriocins (proteins produced by bacteria to kill closely related strains). Despite their differences, the bioactivity of proteins from both systems can be studied in tandem using similar methods: 1) bioinformatic identification of uncharacterized proteins, 2) cloning, expression, and purification of select proteins of interest, and 3) characterization of the purified proteins. By unveiling and analyzing these proteins of great interest, we can gain better access to their function.
11:40 - 12:00 pm
HeLa Cell Toxicity and Cellular DNA-Binding of Rhodium Complexes
Charlotte Reid
Institution: Moravian College
Faculty Mentors: Anastasia Thevenin and Shari Dunham
Cisplatin is a commonly prescribed cancer treatment with harmful side effects to patients and a propensity for chemotherapeutic resistance. Because of these negative aspects of cisplatin, potential anticancer complexes containing rhodium and their effect on human cervical cancer cells have been studied in our laboratories. These dirhodium complexes contain a variety of bridging carboxylate ligands, including acetate, trifluoroacetate, gluconate, and glucuronate. In our studies of the IC50 values of these compounds, the dirhodium complex with four acetate ligands has been the most cytotoxic. DNA of treated cells was also extracted and analyzed for bound rhodium. We continue to characterize these compounds to understand their mechanisms of action and varying levels of toxicity in cancer cells.
12:00 - 12:20 pm
Probing Conformational Changes of E. coli Adenylate Kinase Using a Vibrational Reporter
Angelica Camilo
Institution: Franklin and Marshall College
Faculty Mentors: Christine Phillips-Piro and Scott Brewer
Unnatural amino acids (UAAs) containing vibrational reporter groups such as a nitrile, azide, or nitro have the potential to serve as effective, site-specific probes of local protein environments. Specifically, the UAA 4-cyano-L-phenylalanine (pCNF) contains a nitrile vibrational reporter that is an effective probe of its local solvation environment due to the position and sensitivity of the nitrile symmetric stretch to local hydrogen bonding interactions, in addition to the relative small size of the nitrile group. Here, pCNF was genetically incorporated at multiple distinct sites in the enzyme adenylate kinase (AK), which catalyzes the conversion of two adenylate diphosphate (ADP) molecules to one adenosine monophosphate (AMP) molecule and one adenosine triphosphate (ATP) molecule. Multiple sites were selected to represent a myriad of local solvation states, some of which are predicted to change upon nucleotide binding as indicated by the X-ray crystal structures of apo and nucleotide bound AK. Temperature-dependent IR studies of the UAA containing AK constructs to probe differences between local protein environments around the nitrile group of pCNF in the apo and nucleotide bound constructs will be presented in addition to the protein expression and purification methodology of these constructs.