Biochemistry Festival of Science 2019 - Student Abstracts | St. Lawrence University Biochemistry

Biochemistry Festival of Science 2019 - Student Abstracts

Festival of Science was held Friday, April 26, 2019.

Lisa Kozodoy, Biochemistry, Faculty Sponsors: Nadia Marano and Lorraine Olendzenski
“Isolating amyloid fibers from Haloferax volcanii” 

Abstract: Amyloids are proteins that when aggregated form large insoluble fibers characterized by their cross-beta sheet structure. While these proteins are often implicated in human neurodegenerative diseases, such as Alzheimer’s, many organisms make functional amyloids. 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 amyloid fibers from H. volcanii to elucidate more about their composition and structure. Isolation is done through differential centrifugation and sonication, following previous research by Heather Raimer ‘17. Sonicating the cells releases the amyloid fibers from the cells, which are detected using Thioflavin T (ThT) assays. Low-speed centrifugation allows for the removal of cellular debris. Ultracentrifugation then isolates the larger fibers from suspension. Lastly, tube SDS-PAGE is used to purify the amyloids from contaminating proteins because they do not enter the gel. Current research is being done to deaggregate the fibers into monomers using formic acid. The depolymerized monomers can then be resuspended and sent off to a collaborator for sequencing.


Evan Ketcham, Biochemistry, Faculty Sponsor:  Matthew Skeels
“Engineering Thermodynamically Stable Mutants of Human Basic Fibroblast Growth Factor Protein”

Abstract: 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 mutants known as FGFbase (N71E, E78K, K86E, T112E) and FGFrecp (N53E, K68E, R102E, K111E, K117E) being more stable under chemical denaturing conditions than the wild type FGF2. Further research is being conducted to probe more mutants for their stabilities and assess their relative biological activities using cell cultures.


Lillian Devereux, Biochemistry, Faculty Sponsor Nadia Marano
"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)"

Abstract: 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. ThT is a molecular rotor meaning that it rotates around a single bond. When bound to amyloid fibers this rotation is inhibited and stabilizes the excited state, thus producing measurable fluorescence. However, ThT is not an effective measure of amyloid oligomer formation, the smaller building blocks of mature fibers. ANS, Bis-ANS, and DCVJ are three alternatives to ThT that are characterized by a multi-ring structure. DCVJ acts in a similar way to ThT in that, when bound, the molecule is locked into an excited state producing fluorescence. In contrast, ANS and Bis-ANS bind to the hydrophobic regions of proteins which are exposed during amyloid fiber formation and following binding, the molecules fluoresce. 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. Preliminary research shows DCVJ and ANS follow the pattern of ThT, meaning that as mature fiber concentration increases, so does the fluorescence signal. Bis-ANS, however, displays the opposite pattern, indicating it is likely detecting varying concentrations of amyloid oligomers via binding to the exposed hydrophobic residues.