SYE 2020 Biochemistry

Hannah Bovee - Advisors: Dr. Nadia Marano and Dr. Lorraine Oldenzenski

Characterization of Amyloid Fibers formed by Resistant Soil Bacteria

Amyloid fibers are proteins formed by repeating protein monomers which stack to create a cross-beta sheet quaternary structure. Though the unregulated production of amyloid fibers is often associated with diseases such as Alzheimer’s and Parkinson’s, amyloid fibers are also produced in a regulated manner by bacteria in biofilms to protect against antibiotics and to attach cells to the surface they live on. Previous St. Lawrence researchers isolated a number of antibiotic resistant soil bacteria from a pig farm in upstate New York. Several of these bacteria produced biofilms which tested positive for amyloid fibers with a Thioflavin T (ThT) assay. An isolation procedure utilizing sonication and differential centrifugation to release and separate fibers from Microbacterium isolate #29 was developed by Jack Mechler ’18. This procedure is being adapted to increase the percent recovery of the isolation and improve purity. Two detergents, Triton-100X and hexadecyltrimethylammonium bromide (CTAB), are being assessed as a means of disrupting the cell membranes to separate them from soluble proteins during the procedure. The fluorescence of the detergents below and above their critical micelle concentrations in the presence of ThT was investigated to determine if adding a detergent treatment step would interfere with the ThT assay (as the commonly used detergent sodium dodecyl sulfate does). Additionally, the original sonication treatment is being compared to an extended sonication treatment to assess if more fibers can be released from the cell membrane. Fibers will be purified further using an SDS-PAGE gel and depolymerized using formic acid. The pure amyloid protein monomers will then be sequenced by a collaborator.

Erica Sawyer - Advisor: Dr. Nadia Marano

Isolation and Purification of Amyloid Proteins

Amyloids are a class of aggregated proteins that share a common structural motif, the cross- beta sheet, allowing them to form long fibrils. These fibrils can be pathogenic or can serve many important physiological roles that are not fully understood. Haloferax volcanii, an extremophile species of Archaea, is one microorganism that incorporates functional amyloids into its biofilms; these proteins are proposed to contribute to the structural stability of this matrix. This study aims to improve the efficiency of the isolation and purification of amyloid proteins found in H. volcanii to ultimately allow for further investigation of their structure. Previously, a protocol was established by Lisa Kozodoy ’19 that utilizes differential centrifugation and sonication to isolate amyloid proteins, which are then purified using SDS- PAGE. To improve the effectiveness of this procedure, detergents will be employed during the isolation to disrupt amyloid interactions with the cell membrane, possibly releasing more protein. Additionally, these detergents will prevent the incorporation of Thioflavin T, a fluorescent molecule used to selectively quantify amyloid proteins, into lipid components of the cells that are not removed during the isolation. Triton X-100 and Cetrimonium Bromide, a neutral and cationic detergent, respectively, will be used during the isolation. Then, the yield of pure amyloid proteins will be compared to that obtained without the use of detergents. Purified amyloids will be deaggregated into their monomeric forms using formic acid and resuspended in urea; these proteins can then be sequenced to gain further information regarding their structure.