Brynn earned her B.S. in Biochemistry from Hillsdale College in 2023. Her undergraduate research in the laboratory of Dr. Kelli Kazmier focused on the conformational dynamics of LeuT, an integral membrane protein and model of SERT. In 2021, Brynn also interned at the Van Andel Institute in Grand Rapids, MI in the laboratory of Dr. Connie Krawczyk. While at the Van Andel Institute, Brynn generated MEF clones with HA-tagged KDM5C and KDM5D for use in the Krawczyk Lab's investigation of KDM5C/KDM5D's role in the immune system. Brynn joined the University of Michigan in 2023 to pursue her Ph.D. in Immunology.
Biography
Research Interest
Cancer immunotherapy
Techniques Used
Western blotting, protein purification, HPLC, CW-EPR, DEER EPR, mammalian cell culture, phage display, gel electrophoresis, SDS-PAGE, PCR, flow cytometry
Abstracts
KDM5C is an X-linked demethylase that removes methyl groups from H3K4 (histone 3 lysine 4). KDM5C is most well-known for the role that it plays in X-linked intellectual disability, though mutations in KDM5C are also linked to chemoresistance in pediatric AML. The Krawczyk Laboratory is interested in whether KDM5C and KDM5D exhibit sexual dimorphism. Since antibodies for these genes are not widely available and have low affinity, we generated Mouse Embryonic Fibroblast (MEF) clones with HA-tagged KDM5C/KDM5D. Endogenous KDM5C was removed with the Cre-lox system and replaced with a tagged construct. Successful removal of endogenous KDM5C was verified with a Western blot, and successful knockout clones were isolated with flow cytometry. The Krawczyk Laboratory plans to use these cell lines to perform ChIP-seq to gather information about KDM5C’s genomic location, its regulation of gene expression, and its interactions with other genes. LeuT, an integral membrane protein and member of the NSS family, is a bacterial homolog of human neurotransmitter transporters. The mechanism of LeuT transport has been described by three conformations captured by X-ray crystallography: outward-facing, outward-facing occluded, and inward-facing. To select for an inward-facing conformation, a number of conserved residues were mutated. Using DEER EPR spectroscopy, we have investigated the effects of one set of these mutations, T354 and S355, in LeuT. In this data, we describe the effects of the loss of Na binding on the conformational sampling as well as evaluate whether the crystallographic inward-facing structures represents a natively-sampled LeuT conformation.
Hobbies & Extra Curriculum Activities
Running, playing the clarinet, reading