Biochemistry, Biophysics, and Structural Biology Commons^™

The Study Of The Structure And Dynamics Of Parkin Activation, Elaine Aisha Freeman

Electronic Thesis and Dissertation Repository

Parkin is an RBR E3 ubiquitin ligase that has been implicated in both sporadic and familial Parkinson’s disease. Upon mitochondrial damage, parkin is activated step-wise to recruit and ligate ubiquitin to a substrate on the outer mitochondrial membrane. Disruption of this activation and ligation cascade is hypothesized to result in neuronal death related to Parkinson’s disease.

While structures of parkin for a number of these activation states exist, it is important to note they are not of full-length human parkin. These structures are often truncated and come from various non-human species to eliminate important, yet hard to quantify structural elements. …

Don't Sell Them Short, There's More To Bacterial Natural Products Than Antibiotics, Alison Clare Domzalski

Dissertations, Theses, and Capstone Projects

Recent genomic studies of microbiomes have revealed an overwhelming number of biosynthetic genes of unknown function. Most of these “cryptic” biosynthetic genes are not expressed in laboratory monocultures of individual microbes. Thus, there remains tremendous untapped potential for natural products discovery. Here we employ mixed microbial culture (MMC) as a simple yet powerful approach to awaken cryptic biosynthetic gene clusters. Our preliminary studies demonstrated that arrays of metabolites could be induced in MMCs upon environmental cues, such as surface adhesion. Using this system, we have screened, identified, and isolated bioactive bacterial metabolites, which were characterized structurally and biologically. Of the …

Identification Of Fluoxetine-Sert Interactions And Apo-Sert Studies Via Crosslinking Mass Spectrometry, Elizabeth Castellano

Electronic Theses and Dissertations

The serotonin transporter (SERT) is a member of the neurotransmitter sodium symporter family of transporters. SERT controls the magnitude and duration of serotonergic neurotransmission by facilitating the reuptake of serotonin back into the pre-synaptic neuron and is thus a target for antidepressants. Selective serotonin re-uptake inhibitors (SSRIs) such as fluoxetine, are commonly prescribed to treat depression. SSRIs act by blocking reuptake and prolonging serotonin signaling. However, significant problems regarding selectivity and mechanisms of action of these drugs remain unresolved. The structures of SERT and related transporters have been determined and serve as useful structural models. However, they are typically mutated …

Uncovering The Ubiquitin Ligase Activity And Substrates Of The Human C-Terminal To Lish (Ctlh) Complex, Matthew E.R. Maitland

Electronic Thesis and Dissertation Repository

Ubiquitination is the transfer of a ubiquitin molecule to protein substrates by the sequential actions of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. It is a post-translational modification that controls the fate and function of the substrate protein. Substrate specificity in the ubiquitination reaction is conferred by the E3 ligases. Sequence homology suggests the human C-terminal to LisH (CTLH) complex could be an E3 ligase; however, very little is known about this complex. In this thesis, I characterize the human CTLH complex as a multi-subunit E3 ligase and define its activity, structure, and substrates. I demonstrate that the …

A Workflow To Analyze Ethcd Mass Spectrometry Data For Studying Hiv Gp120 Glycosylation, Yingxue Sun

Electronic Thesis and Dissertation Repository

The great heterogeneity of HIV populations and richness of surface glycan clouds makes it difficult to locate a conserved and exposed protein epitope as an effective vaccine target. However, more than 80% new infections result from single transmitted founder (T/F) viruses. We set out to design a workflow to study the traits of T/Fs that allow for their superior infectivity, specifically, the glycosylation patterns of gp120, a subunit of HIV envelope protein responsible for binding to host cell receptors. Our main research methods include Western blot and mass spectrometry. Our current understanding of the mass spectrometry data indicates that our …

Mass Spectrometry-Based Strategies In Protein Higher Order Structure Analysis: Fundamentals And Applications In Protein-Ligand Interactions, Xiaoran Liu

Arts & Sciences Electronic Theses and Dissertations

Protein ligand interaction is a fundamental question in biology and biochemistry, and many approaches including X-ray crystallography, nuclear magnetic resonance, cryogenic electron microscopy, mass spectroscopy (MS), infrared spectroscopy, circular dichroism, fluorescence spectroscopy and many others have been applied to address this question. Among these techniques, mass spectroscopy has the advantage of high throughput, low sample amount requirement, and mid-to-high spatial resolution. One of the MS-based approaches is protein footprinting, which utilizes labeling reagents to map the solvent accessible surface of the protein of interest thus deliver structural information. Irreversible labeling is represented by covalent labeling and radical labeling, in which …

Validation Of A Deployable Proteomic Assay For The Serological Screening Of Sexual Assault Samples, Catherine O'Sullivan Brown

Electronic Theses and Dissertations

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing …