2019 SYE

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.Detection of Amyloid Oligomers using ANS (1-Anilinophthalene-8-sulfonic acid), Bis-ANS (4,4’ –Dianilino- 1,1’ –binaphthyl -5,5’-disulfonic acid), and DCVJ (4-dicyanovinyl-julolidine)



Lillian Devereux

Advisor: Dr. Nadia Marano



Amyloid fibers are a component of bacterial biofilms that can be isolated through various protocols for analysis. The standard assay uses Thioflavin T (ThT) to identify the presence and quantity of mature amyloid fibers however, it is not an effective measure of amyloid oligomer formation, the smaller building blocks of mature fibers. ANS, Bis-ANS, and DCJV are three alternatives to ThT that are characterized by a multi-ring structure containing a single bond around which the rings are able rotate. However, when these molecules bind to amyloid fibers the rotation is no longer possible, resulting in an increased fluorescence. Little is currently known about the binding and consequent fluorescence of these molecules to oligomers. This research will examine the ability of these three fluorophores to bind to oligomers throughout the aggregation process, giving an indication of kinetics and method of amyloid fiber formation. Insulin will be used because it forms amyloid fibers under well-defined conditions.

Development and Isolation of Stable Fibroblast Growth Factor 2 Proteins



Evan Ketcham

Advisor: Dr. Matthew Skeels



Fibroblast growth factor 2, also known as FGF basic, is a signaling protein that plays a variety of physiological roles in the body. Of particular interest in recent studies is its ability to promote angiogenesis following blood vessel injury. However, it is a labile protein that tends to break down quickly under the stressors of the human body. Previous research has determined, in silico, several rationally-designed mutant proteins that are predicted to be more stable than the wild type. This project seeks to create these mutants and compare their stability in vitro to FGF2. A bacterial expression system is used to create the protein, which is then purified using chromatographic methods. After purification, the linear extrapolation method (Vivian and Callis 2001) is used to experimentally determine the change in Gibbs free energy of unfolding for the protein. In this method, the unfolding of a protein is followed by monitoring intrinsic tryptophan fluorescence as it is exposed to increasing concentrations of chaotrope. These data can be mathematically transformed into an experimental ΔG value. The data have demonstrated that the trend in stability matches the computational model, with the mutant known as FGFbase (N71E, E78K, K86E, T112E) being more stable under chemical denaturing conditions than the wild type FGF2. Further research is being conducted to probe more mutants for their stabilities.

Isolation and Identification of Amyloid Fibers from Archaeon Haloferax volcanii



Lisa Kozodoy

Advisors: Drs. Nadia Marano and Lorraine Olendzinski



Amyloids are proteins that when aggregated form large insoluble fibers characterized by their common cross beta sheet structure. While these proteins are often implicated in human neurodegenerative diseases, such as Alzheimer’s, many organisms make functional amyloids without the toxic side effects. These have been studied in bacterial biofilms and are known to aid in stability and structure. Archaea also form biofilms; however, they have not been researched as extensively. Functional amyloids have been identified in the archaeon Haloferax volcanii. The aim of this study is to isolate and purify these amyloid fibers from H. volcanii to elucidate more about their composition and structure. Isolation is done through differential centrifugation and sonication, following previous research done by Heather Raimer ‘17. Sonicating the cells releases the amyloid fibers from the cells and centrifugation allows for the separation of the amyloids from cellular debris. Ultracentrifugation is done to attempt to isolate the larger amyloid fibers from suspension. Lastly, tube SDS-PAGE is used to purify the amyloids from any proteins that are contaminating the sample. The amyloid themselves are detected using Thioflavin T (ThT) assays. ThT will have an emission peak at 487 nm when excited and bound to amyloid fibers. The isolated tube gel sample can be deaggregated into its monomers using formic acid. The monomers are resuspended in urea in order to prevent reaggregation. These can then be sent off to a collaborator for sequencing. 

Characterizing the Binding Mechanisms of New Chemotherapy Drugs with Plasmid DNA



So Min (Kate) Park

Advisor: Samantha Glazier



Even with the great amount of research currently underway on cancer treatments, development of new chemotherapy drugs remains one of the biggest challenges in the medical field. While there are a few well-known chemotherapy drugs, such as doxorubicin, further understanding of the binding mechanisms of the drug molecules to DNA is essential for the development of new chemotherapy drugs. Our lab previously characterized the binding mode of doxorubicinone, a doxorubicin derivative, to calf thymus DNA using a melting protocol based on UV-Vis spectrometry and observing whether there were changes in the melting temperatures; intercalators increase the melting temperature of DNA and groove binders do not change the melting temperature of DNA. However, as CT DNA has a mixture of both double and single stranded DNA and has unknown DNA fragment sizes, we believe that a more homogenous mixture of DNA could improve our results. In this project, we will develop a protocol to determine the binding mechanisms of new chemotherapy drugs to plasmid DNA. Unlike the CT DNA that was used previously, plasmid DNA only contains circular double stranded DNA that can be easily linearized with restriction enzymes and has a known DNA sequence length of 2686 base pairs. With a more homogenous mixture of DNA fragments, we will be able to compare melting data collected from linearized plasmid to melting data collected from CT DNA. Because testing with millileter volumes can be expensive, we will develop a low volume protocol that detects changes in fluorescence in a PCR (polymerized chain reaction) instrument to collect melting points. As in the UV-Vis based studies, we can determine the binding mechanisms of chemotherapy drugs with plasmid DNA in a more cost effective way. In both techniques, we hope to obtain sharper inflection points in the melting curves and more distinct first derivative curves, so that these protocols can be used to determine the binding mechanisms of future chemotherapy drugs synthesized in Dr. Tartakoff’s lab. Moreover, if we are successful in improving our melting data with these protocols, we will develop another low volume protocol for determining the binding mechanisms of drugs using an agarose gel, as intercalators cause DNA to recoil and recoiled DNA travels further on a gel than DNA bound to drugs that via a minor groove mode.